GLDS-120 Analysis Using iDEP 0.91

Analysis of GLDS-120 from NASA GeneLab

This R markdown file was auto-generated by the iDEP website Using iDEP 0.91, originally by Steven Xijin.Ge@sdstate.edu

Ge SX, Son EW, Yao R: iDEP: an integrated web application for differential expression and pathway analysis of RNA-Seq data. BMC Bioinformatics 2018, 19(1):534. PMID:30567491

1. Read data

First we set up the working directory to where the files are saved.

 setwd('~/Documents/HTML_R/GLDS120')

R packages and iDEP core Functions. Users can also download the iDEP_core_functions.R file. Many R packages needs to be installed first. This may take hours. Each of these packages took years to develop.So be a patient thief. Sometimes dependencies needs to be installed manually. If you are using an older version of R, and having trouble with package installation, try un-install the current version of R, delete all folders and files (C:/Program Files/R/R-3.4.3), and reinstall from scratch.

 if(file.exists('iDEP_core_functions.R'))
    source('iDEP_core_functions.R') else 
    source('https://raw.githubusercontent.com/iDEP-SDSU/idep/master/shinyapps/idep/iDEP_core_functions.R') 

We are using the downloaded gene expression file where gene IDs has been converted to Ensembl gene IDs. This is because the ID conversion database is too large to download. You can use your original file if your file uses Ensembl ID, or you do not want to use the pathway files available in iDEP (or it is not available).

 inputFile <- 'GLDS120_Expression.csv'
 sampleInfoFile <- 'GLDS120_Sampleinfo.csv'
 gldsMetadataFile <- 'GLDS120_Metadata.csv'
 geneInfoFile <- 'Arabidopsis_thaliana__athaliana_eg_gene_GeneInfo.csv' #Gene symbols, location etc. 
 geneSetFile <- 'Arabidopsis_thaliana__athaliana_eg_gene.db'  # pathway database in SQL; can be GMT format 
 STRING10_speciesFile <- 'https://raw.githubusercontent.com/iDEP-SDSU/idep/master/shinyapps/idep/STRING10_species.csv' 

Parameters for reading data

 input_missingValue <- 'geneMedian' #Missing values imputation method
 input_dataFileFormat <- 1  #1- read counts, 2 FKPM/RPKM or DNA microarray
 input_minCounts <- 0.5 #Min counts
 input_NminSamples <- 1 #Minimum number of samples 
 input_countsLogStart <- 4  #Pseudo count for log CPM
 input_CountsTransform <- 1 #Methods for data transformation of counts. 1-EdgeR's logCPM 2-VST, 3-rlog 

readMetadata.out <- readMetadata(gldsMetadataFile)
library(knitr)   #  install if needed. for showing tables with kable
library(kableExtra)
kable( readMetadata.out ) %>%
  kable_styling(bootstrap_options = c("striped", "hover")) %>%
  scroll_box(width = "100%")

	Col0_GC_Alight_Rep1	Col0_GC_Alight_Rep2	Col0_GC_Alight_Rep3	Col0_GC_dark_Rep1	Col0_GC_dark_Rep2	Col0_GC_dark_Rep3	Ws_GC_Alight_Rep1	Ws_GC_Alight_Rep2	Ws_GC_Alight_Rep3	Ws_GC_dark_Rep1	Ws_GC_dark_Rep2	Ws_GC_dark_Rep3	Col0PhyD_GC_Alight_Rep1	Col0PhyD_GC_Alight_Rep2	Col0PhyD_GC_Alight_Rep3	Col0PhyD_GC_dark_Rep1	Col0PhyD_GC_dark_Rep2	Col0PhyD_GC_dark_Rep3	Col0_FLT_Alight_Rep1	Col0_FLT_Alight_Rep2	Col0_FLT_Alight_Rep3	Col0_FLT_dark_Rep1	Col0_FLT_dark_Rep2	Col0_FLT_dark_Rep3	Ws_FLT_Alight_Rep1	Ws_FLT_Alight_Rep2	Ws_FLT_Alight_Rep3	Ws_FLT_dark_Rep1	Ws_FLT_dark_Rep2	Ws_FLT_dark_Rep3	Col0PhyD_FLT_Alight_Rep1	Col0PhyD_FLT_Alight_Rep2	Col0PhyD_FLT_Alight_Rep3	Col0PhyD_FLT_dark_Rep1	Col0PhyD_FLT_dark_Rep2	Col0PhyD_FLT_dark_Rep3
Sample.LongId	Atha.Col.0.root.GC.Alight.Rep1.GSM2493759.RNAseq.RNAseq	Atha.Col.0.root.GC.Alight.Rep2.GSM2493760.RNAseq.RNAseq	Atha.Col.0.root.GC.Alight.Rep3.GSM2493761.RNAseq.RNAseq	Atha.Col.0.root.GC.dark.Rep1.GSM2493768.RNAseq.RNAseq	Atha.Col.0.root.GC.dark.Rep2.GSM2493769.RNAseq.RNAseq	Atha.Col.0.root.GC.dark.Rep3.GSM2493770.RNAseq.RNAseq	Atha.Ws.root.GC.Alight.Rep1.GSM2493762.RNAseq.RNAseq	Atha.Ws.root.GC.Alight.Rep2.GSM2493763.RNAseq.RNAseq	Atha.Ws.root.GC.Alight.Rep3.GSM2493764.RNAseq.RNAseq	Atha.Ws.root.GC.dark.Rep1.GSM2493771.RNAseq.RNAseq	Atha.Ws.root.GC.dark.Rep2.GSM2493772.RNAseq.RNAseq	Atha.Ws.root.GC.dark.Rep3.GSM2493773.RNAseq.RNAseq	Atha.Col.0.PhyD.root.GC.Alight.Rep1.GSM2493765.RNAseq.RNAseq	Atha.Col.0.PhyD.root.GC.Alight.Rep2.GSM2493766.RNAseq.RNAseq	Atha.Col.0.PhyD.root.GC.Alight.Rep3.GSM2493767.RNAseq.RNAseq	Atha.Col.0.PhyD.root.GC.dark.Rep1.GSM2493774.RNAseq.RNAseq	Atha.Col.0.PhyD.root.GC.dark.Rep2.GSM2493775.RNAseq.RNAseq	Atha.Col.0.PhyD.root.GC.dark.Rep3.GSM2493776.RNAseq.RNAseq	Atha.Col.0.root.FLT.Alight.Rep1.GSM2493777.RNAseq.RNAseq	Atha.Col.0.root.FLT.Alight.Rep2.GSM2493778.RNAseq.RNAseq	Atha.Col.0.root.FLT.Alight.Rep3.GSM2493779.RNAseq.RNAseq	Atha.Col.0.root.FLT.dark.Rep1.GSM2493786.RNAseq.RNAseq	Atha.Col.0.root.FLT.dark.Rep2.GSM2493787.RNAseq.RNAseq	Atha.Col.0.root.FLT.dark.Rep3.GSM2493788.RNAseq.RNAseq	Atha.Ws.root.FLT.Alight.Rep1.GSM2493780.RNAseq.RNAseq	Atha.Ws.root.FLT.Alight.Rep2.GSM2493781.RNAseq.RNAseq	Atha.Ws.root.FLT.Alight.Rep3.GSM2493782.RNAseq.RNAseq	Atha.Ws.root.FLT.dark.Rep1.GSM2493789.RNAseq.RNAseq	Atha.Ws.root.FLT.dark.Rep2.GSM2493790.RNAseq.RNAseq	Atha.Ws.root.FLT.dark.Rep3.GSM2493791.RNAseq.RNAseq	Atha.Col.0.PhyD.root.FLT.Alight.Rep1.GSM2493783.RNAseq.RNAseq	Atha.Col.0.PhyD.root.FLT.Alight.Rep2.GSM2493784.RNAseq.RNAseq	Atha.Col.0.PhyD.root.FLT.Alight.Rep3.GSM2493785.RNAseq.RNAseq	Atha.Col.0.PhyD.root.FLT.dark.Rep1.GSM2493792.RNAseq.RNAseq	Atha.Col.0.PhyD.root.FLT.dark.Rep2.GSM2493793.RNAseq.RNAseq	Atha.Col.0.PhyD.root.FLT.dark.Rep3.GSM2493794.RNAseq.RNAseq
Sample.Id
Sample.Name	Atha_Col-0_root_GC_Alight_Rep1_GSM2493759	Atha_Col-0_root_GC_Alight_Rep2_GSM2493760	Atha_Col-0_root_GC_Alight_Rep3_GSM2493761	Atha_Col-0_root_GC_dark_Rep1_GSM2493768	Atha_Col-0_root_GC_dark_Rep2_GSM2493769	Atha_Col-0_root_GC_dark_Rep3_GSM2493770	Atha_Ws_root_GC_Alight_Rep1_GSM2493762	Atha_Ws_root_GC_Alight_Rep2_GSM2493763	Atha_Ws_root_GC_Alight_Rep3_GSM2493764	Atha_Ws_root_GC_dark_Rep1_GSM2493771	Atha_Ws_root_GC_dark_Rep2_GSM2493772	Atha_Ws_root_GC_dark_Rep3_GSM2493773	Atha_Col-0-PhyD_root_GC_Alight_Rep1_GSM2493765	Atha_Col-0-PhyD_root_GC_Alight_Rep2_GSM2493766	Atha_Col-0-PhyD_root_GC_Alight_Rep3_GSM2493767	Atha_Col-0-PhyD_root_GC_dark_Rep1_GSM2493774	Atha_Col-0-PhyD_root_GC_dark_Rep2_GSM2493775	Atha_Col-0-PhyD_root_GC_dark_Rep3_GSM2493776	Atha_Col-0_root_FLT_Alight_Rep1_GSM2493777	Atha_Col-0_root_FLT_Alight_Rep2_GSM2493778	Atha_Col-0_root_FLT_Alight_Rep3_GSM2493779	Atha_Col-0_root_FLT_dark_Rep1_GSM2493786	Atha_Col-0_root_FLT_dark_Rep2_GSM2493787	Atha_Col-0_root_FLT_dark_Rep3_GSM2493788	Atha_Ws_root_FLT_Alight_Rep1_GSM2493780	Atha_Ws_root_FLT_Alight_Rep2_GSM2493781	Atha_Ws_root_FLT_Alight_Rep3_GSM2493782	Atha_Ws_root_FLT_dark_Rep1_GSM2493789	Atha_Ws_root_FLT_dark_Rep2_GSM2493790	Atha_Ws_root_FLT_dark_Rep3_GSM2493791	Atha_Col-0-PhyD_root_FLT_Alight_Rep1_GSM2493783	Atha_Col-0-PhyD_root_FLT_Alight_Rep2_GSM2493784	Atha_Col-0-PhyD_root_FLT_Alight_Rep3_GSM2493785	Atha_Col-0-PhyD_root_FLT_dark_Rep1_GSM2493792	Atha_Col-0-PhyD_root_FLT_dark_Rep2_GSM2493793	Atha_Col-0-PhyD_root_FLT_dark_Rep3_GSM2493794
GLDS	120	120	120	120	120	120	120	120	120	120	120	120	120	120	120	120	120	120	120	120	120	120	120	120	120	120	120	120	120	120	120	120	120	120	120	120
Accession	GLDS-120	GLDS-120	GLDS-120	GLDS-120	GLDS-120	GLDS-120	GLDS-120	GLDS-120	GLDS-120	GLDS-120	GLDS-120	GLDS-120	GLDS-120	GLDS-120	GLDS-120	GLDS-120	GLDS-120	GLDS-120	GLDS-120	GLDS-120	GLDS-120	GLDS-120	GLDS-120	GLDS-120	GLDS-120	GLDS-120	GLDS-120	GLDS-120	GLDS-120	GLDS-120	GLDS-120	GLDS-120	GLDS-120	GLDS-120	GLDS-120	GLDS-120
Hardware	Petri dish	Petri dish	Petri dish	Petri dish	Petri dish	Petri dish	Petri dish	Petri dish	Petri dish	Petri dish	Petri dish	Petri dish	Petri dish	Petri dish	Petri dish	Petri dish	Petri dish	Petri dish	Petri dish	Petri dish	Petri dish	Petri dish	Petri dish	Petri dish	Petri dish	Petri dish	Petri dish	Petri dish	Petri dish	Petri dish	Petri dish	Petri dish	Petri dish	Petri dish	Petri dish	Petri dish
Tissue	Roots	Roots	Roots	Roots	Roots	Roots	Roots	Roots	Roots	Roots	Roots	Roots	Roots	Roots	Roots	Roots	Roots	Roots	Roots	Roots	Roots	Roots	Roots	Roots	Roots	Roots	Roots	Roots	Roots	Roots	Roots	Roots	Roots	Roots	Roots	Roots
Age	11 days	11 days	11 days	11 days	11 days	11 days	11 days	11 days	11 days	11 days	11 days	11 days	11 days	11 days	11 days	11 days	11 days	11 days	11 days	11 days	11 days	11 days	11 days	11 days	11 days	11 days	11 days	11 days	11 days	11 days	11 days	11 days	11 days	11 days	11 days	11 days
Organism	Arabidopsis thaliana	Arabidopsis thaliana	Arabidopsis thaliana	Arabidopsis thaliana	Arabidopsis thaliana	Arabidopsis thaliana	Arabidopsis thaliana	Arabidopsis thaliana	Arabidopsis thaliana	Arabidopsis thaliana	Arabidopsis thaliana	Arabidopsis thaliana	Arabidopsis thaliana	Arabidopsis thaliana	Arabidopsis thaliana	Arabidopsis thaliana	Arabidopsis thaliana	Arabidopsis thaliana	Arabidopsis thaliana	Arabidopsis thaliana	Arabidopsis thaliana	Arabidopsis thaliana	Arabidopsis thaliana	Arabidopsis thaliana	Arabidopsis thaliana	Arabidopsis thaliana	Arabidopsis thaliana	Arabidopsis thaliana	Arabidopsis thaliana	Arabidopsis thaliana	Arabidopsis thaliana	Arabidopsis thaliana	Arabidopsis thaliana	Arabidopsis thaliana	Arabidopsis thaliana	Arabidopsis thaliana
Ecotype	Col-0	Col-0	Col-0	Col-0	Col-0	Col-0	WS-0	WS-0	WS-0	WS-0	WS-0	WS-0	Col-0	Col-0	Col-0	Col-0	Col-0	Col-0	Col-0	Col-0	Col-0	Col-0	Col-0	Col-0	WS-0	WS-0	WS-0	WS-0	WS-0	WS-0	Col-0	Col-0	Col-0	Col-0	Col-0	Col-0
Genotype	WT	WT	WT	WT	WT	WT	WT	WT	WT	WT	WT	WT	PhyD	PhyD	PhyD	PhyD	PhyD	PhyD	WT	WT	WT	WT	WT	WT	WT	WT	WT	WT	WT	WT	PhyD	PhyD	PhyD	PhyD	PhyD	PhyD
Variety	Col-0 WT	Col-0 WT	Col-0 WT	Col-0 WT	Col-0 WT	Col-0 WT	WS-0 WT	WS-0 WT	WS-0 WT	WS-0 WT	WS-0 WT	WS-0 WT	Col-0 PhyD	Col-0 PhyD	Col-0 PhyD	Col-0 PhyD	Col-0 PhyD	Col-0 PhyD	Col-0 WT	Col-0 WT	Col-0 WT	Col-0 WT	Col-0 WT	Col-0 WT	WS-0 WT	WS-0 WT	WS-0 WT	WS-0 WT	WS-0 WT	WS-0 WT	Col-0 PhyD	Col-0 PhyD	Col-0 PhyD	Col-0 PhyD	Col-0 PhyD	Col-0 PhyD
Radiation	Background Earth	Background Earth	Background Earth	Background Earth	Background Earth	Background Earth	Background Earth	Background Earth	Background Earth	Background Earth	Background Earth	Background Earth	Background Earth	Background Earth	Background Earth	Background Earth	Background Earth	Background Earth	Cosmic radiation	Cosmic radiation	Cosmic radiation	Cosmic radiation	Cosmic radiation	Cosmic radiation	Cosmic radiation	Cosmic radiation	Cosmic radiation	Cosmic radiation	Cosmic radiation	Cosmic radiation	Cosmic radiation	Cosmic radiation	Cosmic radiation	Cosmic radiation	Cosmic radiation	Cosmic radiation
Gravity	Terrestrial	Terrestrial	Terrestrial	Terrestrial	Terrestrial	Terrestrial	Terrestrial	Terrestrial	Terrestrial	Terrestrial	Terrestrial	Terrestrial	Terrestrial	Terrestrial	Terrestrial	Terrestrial	Terrestrial	Terrestrial	Microgravity	Microgravity	Microgravity	Microgravity	Microgravity	Microgravity	Microgravity	Microgravity	Microgravity	Microgravity	Microgravity	Microgravity	Microgravity	Microgravity	Microgravity	Microgravity	Microgravity	Microgravity
Developmental	11 day old seedling roots	11 day old seedling roots	11 day old seedling roots	11 day old seedling roots	11 day old seedling roots	11 day old seedling roots	11 day old seedling roots	11 day old seedling roots	11 day old seedling roots	11 day old seedling roots	11 day old seedling roots	11 day old seedling roots	11 day old seedling roots	11 day old seedling roots	11 day old seedling roots	11 day old seedling roots	11 day old seedling roots	11 day old seedling roots	11 day old seedling roots	11 day old seedling roots	11 day old seedling roots	11 day old seedling roots	11 day old seedling roots	11 day old seedling roots	11 day old seedling roots	11 day old seedling roots	11 day old seedling roots	11 day old seedling roots	11 day old seedling roots	11 day old seedling roots	11 day old seedling roots	11 day old seedling roots	11 day old seedling roots	11 day old seedling roots	11 day old seedling roots	11 day old seedling roots
Time.series.or.Concentration.gradient	Single time point	Single time point	Single time point	Single time point	Single time point	Single time point	Single time point	Single time point	Single time point	Single time point	Single time point	Single time point	Single time point	Single time point	Single time point	Single time point	Single time point	Single time point	Single time point	Single time point	Single time point	Single time point	Single time point	Single time point	Single time point	Single time point	Single time point	Single time point	Single time point	Single time point	Single time point	Single time point	Single time point	Single time point	Single time point	Single time point
Light	4-6 umoles m-2 s-1 total light	4-6 umoles m-2 s-1 total light	4-6 umoles m-2 s-1 total light	Light-activated for 4 hours, then dark-grown	Light-activated for 4 hours, then dark-grown	Light-activated for 4 hours, then dark-grown	4-6 umoles m-2 s-1 total light	4-6 umoles m-2 s-1 total light	4-6 umoles m-2 s-1 total light	Light-activated for 4 hours, then dark-grown	Light-activated for 4 hours, then dark-grown	Light-activated for 4 hours, then dark-grown	4-6 umoles m-2 s-1 total light	4-6 umoles m-2 s-1 total light	4-6 umoles m-2 s-1 total light	Light-activated for 4 hours, then dark-grown	Light-activated for 4 hours, then dark-grown	Light-activated for 4 hours, then dark-grown	4-6 umoles m-2 s-1 total light	4-6 umoles m-2 s-1 total light	4-6 umoles m-2 s-1 total light	Light-activated for 4 hours, then dark-grown	Light-activated for 4 hours, then dark-grown	Light-activated for 4 hours, then dark-grown	4-6 umoles m-2 s-1 total light	4-6 umoles m-2 s-1 total light	4-6 umoles m-2 s-1 total light	Light-activated for 4 hours, then dark-grown	Light-activated for 4 hours, then dark-grown	Light-activated for 4 hours, then dark-grown	4-6 umoles m-2 s-1 total light	4-6 umoles m-2 s-1 total light	4-6 umoles m-2 s-1 total light	Light-activated for 4 hours, then dark-grown	Light-activated for 4 hours, then dark-grown	Light-activated for 4 hours, then dark-grown
Analysis.Light	Alight	Alight	Alight	Dark	Dark	Dark	Alight	Alight	Alight	Dark	Dark	Dark	Alight	Alight	Alight	Dark	Dark	Dark	Alight	Alight	Alight	Dark	Dark	Dark	Alight	Alight	Alight	Dark	Dark	Dark	Alight	Alight	Alight	Dark	Dark	Dark
Assay..RNAseq.	RNAseq Transcription Profiling	RNAseq Transcription Profiling	RNAseq Transcription Profiling	RNAseq Transcription Profiling	RNAseq Transcription Profiling	RNAseq Transcription Profiling	RNAseq Transcription Profiling	RNAseq Transcription Profiling	RNAseq Transcription Profiling	RNAseq Transcription Profiling	RNAseq Transcription Profiling	RNAseq Transcription Profiling	RNAseq Transcription Profiling	RNAseq Transcription Profiling	RNAseq Transcription Profiling	RNAseq Transcription Profiling	RNAseq Transcription Profiling	RNAseq Transcription Profiling	RNAseq Transcription Profiling	RNAseq Transcription Profiling	RNAseq Transcription Profiling	RNAseq Transcription Profiling	RNAseq Transcription Profiling	RNAseq Transcription Profiling	RNAseq Transcription Profiling	RNAseq Transcription Profiling	RNAseq Transcription Profiling	RNAseq Transcription Profiling	RNAseq Transcription Profiling	RNAseq Transcription Profiling	RNAseq Transcription Profiling	RNAseq Transcription Profiling	RNAseq Transcription Profiling	RNAseq Transcription Profiling	RNAseq Transcription Profiling	RNAseq Transcription Profiling
Temperature
Treatment.type
Treatment.intensity	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X
Treament.timing	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X	X
Preservation.Method.	RNAlater	RNAlater	RNAlater	RNAlater	RNAlater	RNAlater	RNAlater	RNAlater	RNAlater	RNAlater	RNAlater	RNAlater	RNAlater	RNAlater	RNAlater	RNAlater	RNAlater	RNAlater	RNAlater	RNAlater	RNAlater	RNAlater	RNAlater	RNAlater	RNAlater	RNAlater	RNAlater	RNAlater	RNAlater	RNAlater	RNAlater	RNAlater	RNAlater	RNAlater	RNAlater	RNAlater

 readData.out <- readData(inputFile) 

## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors

   kable( head(readData.out$data) ) %>%
  kable_styling(bootstrap_options = c("striped", "hover")) %>%
  scroll_box(width = "100%") 

	Col0_GC_Alight_Rep1	Col0_GC_Alight_Rep2	Col0_GC_Alight_Rep3	Col0_GC_dark_Rep1	Col0_GC_dark_Rep2	Col0_GC_dark_Rep3	Ws_GC_Alight_Rep1	Ws_GC_Alight_Rep2	Ws_GC_Alight_Rep3	Ws_GC_dark_Rep1	Ws_GC_dark_Rep2	Ws_GC_dark_Rep3	Col0PhyD_GC_Alight_Rep1	Col0PhyD_GC_Alight_Rep2	Col0PhyD_GC_Alight_Rep3	Col0PhyD_GC_dark_Rep1	Col0PhyD_GC_dark_Rep2	Col0PhyD_GC_dark_Rep3	Col0_FLT_Alight_Rep1	Col0_FLT_Alight_Rep2	Col0_FLT_Alight_Rep3	Col0_FLT_dark_Rep1	Col0_FLT_dark_Rep2	Col0_FLT_dark_Rep3	Ws_FLT_Alight_Rep1	Ws_FLT_Alight_Rep2	Ws_FLT_Alight_Rep3	Ws_FLT_dark_Rep1	Ws_FLT_dark_Rep2	Ws_FLT_dark_Rep3	Col0PhyD_FLT_Alight_Rep1	Col0PhyD_FLT_Alight_Rep2	Col0PhyD_FLT_Alight_Rep3	Col0PhyD_FLT_dark_Rep1	Col0PhyD_FLT_dark_Rep2	Col0PhyD_FLT_dark_Rep3
AT1G07590	14.09484	15.82643	16.00050	13.10404	13.51777	14.64386	13.409439	15.77315	14.82674	8.734572	12.853189	14.49060	14.30307	16.42429	16.25823	14.93434	14.023780	13.652232	13.06820	13.47644	15.85437	14.53257	12.546514	16.29378	13.70761	17.15691	15.84185	11.771773	15.17731	15.44411	15.63005	16.46416	16.53664	16.16638	14.22419	11.227882
AT1G21310	13.82237	12.89321	13.59194	12.69178	11.84638	13.57377	11.609280	14.87147	13.40320	10.803775	11.668910	12.93797	12.21651	15.52722	14.64023	14.21443	13.702426	13.487243	13.86935	13.89505	14.02481	14.15226	13.295761	15.67999	14.01774	15.40720	14.53493	13.029378	14.92947	14.70538	14.61414	13.92094	14.68044	14.58744	13.84959	11.987155
AT2G33830	13.00360	12.70793	13.19989	11.07625	11.44428	12.10500	11.605670	13.01332	13.23629	6.342765	8.041026	10.62547	13.12869	14.63728	13.86356	12.00093	9.571015	8.582187	13.84084	13.54377	14.43678	13.47765	11.086524	14.73928	13.32348	14.63064	13.90582	9.399696	12.33273	12.11427	14.45015	14.83492	14.74970	13.46973	13.62021	13.043726
AT1G07610	12.08104	14.30673	14.23944	10.40031	11.39977	12.30921	9.268934	12.67998	11.15489	4.495592	9.733672	10.63264	11.22988	14.58783	14.11140	12.85493	11.888352	11.502633	10.64115	11.46993	13.77730	12.79971	9.080162	14.60663	10.27736	13.91128	12.58584	8.510065	11.83719	12.25284	13.89512	14.26232	14.34338	13.98226	11.39071	6.943481
AT5G03240	13.20130	13.12291	13.32156	11.49666	11.57663	13.03192	12.702830	13.73541	14.16480	10.261950	10.374225	11.62274	13.07120	14.91590	14.53655	13.45543	11.608011	11.671690	12.73701	13.21551	13.74191	12.73065	10.975705	12.96365	12.31093	14.01545	13.06204	10.566490	12.53392	12.64681	13.26962	14.37130	14.68130	13.65752	12.14785	12.731019
AT2G45960	14.76921	14.16179	14.40573	12.98223	12.07059	13.29898	13.412024	14.52394	14.19108	11.907155	12.186793	12.66575	12.72364	15.00718	14.16692	13.92458	12.483819	12.222020	13.90170	13.63596	14.27782	13.88254	12.522675	13.61255	14.13872	13.88720	14.02430	11.971582	13.47262	13.65144	13.90907	13.79361	14.08793	13.30008	12.89654	12.072701

 readSampleInfo.out <- readSampleInfo(sampleInfoFile) 
 kable( readSampleInfo.out ) %>%
  kable_styling(bootstrap_options = c("striped", "hover")) %>%
  scroll_box(width = "100%") 

	Gravity	Variety	Light
Col0_GC_Alight_Rep1	Terrestrial	Col0 WT	Alight
Col0_GC_Alight_Rep2	Terrestrial	Col0 WT	Alight
Col0_GC_Alight_Rep3	Terrestrial	Col0 WT	Alight
Col0_GC_dark_Rep1	Terrestrial	Col0 WT	Dark
Col0_GC_dark_Rep2	Terrestrial	Col0 WT	Dark
Col0_GC_dark_Rep3	Terrestrial	Col0 WT	Dark
Ws_GC_Alight_Rep1	Terrestrial	WS0 WT	Alight
Ws_GC_Alight_Rep2	Terrestrial	WS0 WT	Alight
Ws_GC_Alight_Rep3	Terrestrial	WS0 WT	Alight
Ws_GC_dark_Rep1	Terrestrial	WS0 WT	Dark
Ws_GC_dark_Rep2	Terrestrial	WS0 WT	Dark
Ws_GC_dark_Rep3	Terrestrial	WS0 WT	Dark
Col0PhyD_GC_Alight_Rep1	Terrestrial	Col0 PhyD	Alight
Col0PhyD_GC_Alight_Rep2	Terrestrial	Col0 PhyD	Alight
Col0PhyD_GC_Alight_Rep3	Terrestrial	Col0 PhyD	Alight
Col0PhyD_GC_dark_Rep1	Terrestrial	Col0 PhyD	Dark
Col0PhyD_GC_dark_Rep2	Terrestrial	Col0 PhyD	Dark
Col0PhyD_GC_dark_Rep3	Terrestrial	Col0 PhyD	Dark
Col0_FLT_Alight_Rep1	Microgravity	Col0 WT	Alight
Col0_FLT_Alight_Rep2	Microgravity	Col0 WT	Alight
Col0_FLT_Alight_Rep3	Microgravity	Col0 WT	Alight
Col0_FLT_dark_Rep1	Microgravity	Col0 WT	Dark
Col0_FLT_dark_Rep2	Microgravity	Col0 WT	Dark
Col0_FLT_dark_Rep3	Microgravity	Col0 WT	Dark
Ws_FLT_Alight_Rep1	Microgravity	WS0 WT	Alight
Ws_FLT_Alight_Rep2	Microgravity	WS0 WT	Alight
Ws_FLT_Alight_Rep3	Microgravity	WS0 WT	Alight
Ws_FLT_dark_Rep1	Microgravity	WS0 WT	Dark
Ws_FLT_dark_Rep2	Microgravity	WS0 WT	Dark
Ws_FLT_dark_Rep3	Microgravity	WS0 WT	Dark
Col0PhyD_FLT_Alight_Rep1	Microgravity	Col0 PhyD	Alight
Col0PhyD_FLT_Alight_Rep2	Microgravity	Col0 PhyD	Alight
Col0PhyD_FLT_Alight_Rep3	Microgravity	Col0 PhyD	Alight
Col0PhyD_FLT_dark_Rep1	Microgravity	Col0 PhyD	Dark
Col0PhyD_FLT_dark_Rep2	Microgravity	Col0 PhyD	Dark
Col0PhyD_FLT_dark_Rep3	Microgravity	Col0 PhyD	Dark

 input_selectOrg ="NEW" 
 input_selectGO <- 'GOBP'   #Gene set category 
 input_noIDConversion = TRUE  
 allGeneInfo.out <- geneInfo(geneInfoFile) 
 converted.out = NULL 
 convertedData.out <- convertedData()    
 nGenesFilter()  

## [1] "16156 genes in 36 samples. 16155  genes passed filter.\n Original gene IDs used."

 convertedCounts.out <- convertedCounts()  # converted counts, just for compatibility 

2. Pre-process

# Read counts per library 
 parDefault = par() 
 par(mar=c(12,4,2,2)) 
 # barplot of total read counts
 x <- readData.out$rawCounts
 groups = as.factor( detectGroups(colnames(x ) ) )
 if(nlevels(groups)<=1 | nlevels(groups) >20 )  
  col1 = 'green'  else
  col1 = rainbow(nlevels(groups))[ groups ]             
         
 barplot( colSums(x)/1e6, 
        col=col1,las=3, main="Total read counts (millions)")  

 readCountsBias()  # detecting bias in sequencing depth 

## [1] 0.05123677
## [1] 0.5460606
## [1] 0.2013552
## [1] 0.3019591

## [1] "No bias detected"

 # Box plot 
 x = readData.out$data 
 boxplot(x, las = 2, col=col1,
    ylab='Transformed expression levels',
    main='Distribution of transformed data') 

 #Density plot 
 par(parDefault) 

## Warning in par(parDefault): graphical parameter "cin" cannot be set

## Warning in par(parDefault): graphical parameter "cra" cannot be set

## Warning in par(parDefault): graphical parameter "csi" cannot be set

## Warning in par(parDefault): graphical parameter "cxy" cannot be set

## Warning in par(parDefault): graphical parameter "din" cannot be set

## Warning in par(parDefault): graphical parameter "page" cannot be set

 densityPlot()       

 # Scatter plot of the first two samples 
 plot(x[,1:2],xlab=colnames(x)[1],ylab=colnames(x)[2], 
    main='Scatter plot of first two samples') 

 ####plot gene or gene family
 input_selectOrg ="BestMatch" 
 input_geneSearch <- 'HOXA' #Gene ID for searching 
 genePlot()  

## NULL

 input_useSD <- 'FALSE' #Use standard deviation instead of standard error in error bar? 
 geneBarPlotError()       

## NULL

3. Heatmap

 # hierarchical clustering tree
 x <- readData.out$data
 maxGene <- apply(x,1,max)
 # remove bottom 25% lowly expressed genes, which inflate the PPC
 x <- x[which(maxGene > quantile(maxGene)[1] ) ,] 
 plot(as.dendrogram(hclust2( dist2(t(x)))), ylab="1 - Pearson C.C.", type = "rectangle") 

 #Correlation matrix
 input_labelPCC <- TRUE #Show correlation coefficient? 
 correlationMatrix() 

 # Parameters for heatmap
 input_nGenes <- 1000   #Top genes for heatmap
 input_geneCentering <- TRUE    #centering genes ?
 input_sampleCentering <- FALSE #Center by sample?
 input_geneNormalize <- FALSE   #Normalize by gene?
 input_sampleNormalize <- FALSE #Normalize by sample?
 input_noSampleClustering <- FALSE  #Use original sample order
 input_heatmapCutoff <- 4   #Remove outliers beyond number of SDs 
 input_distFunctions <- 1   #which distant funciton to use
 input_hclustFunctions <- 1 #Linkage type
 input_heatColors1 <- 1 #Colors
 input_selectFactorsHeatmap <- 'Light'  #Sample coloring factors 
 png('heatmap.png', width = 10, height = 15, units = 'in', res = 300) 
 staticHeatmap() 
 dev.off()  

## png 
##   2

[heatmap] (heatmap.png)

 heatmapPlotly() # interactive heatmap using Plotly 

4. K-means clustering

 input_nGenesKNN <- 2000    #Number of genes fro k-Means
 input_nClusters <- 4   #Number of clusters 
 maxGeneClustering = 12000
 input_kmeansNormalization <- 'geneMean'    #Normalization
 input_KmeansReRun <- 0 #Random seed 

 distributionSD()  #Distribution of standard deviations 

 KmeansNclusters()  #Number of clusters 

 Kmeans.out = Kmeans()   #Running K-means 
 KmeansHeatmap()   #Heatmap for k-Means 

 #Read gene sets for enrichment analysis 
 sqlite  <- dbDriver('SQLite')
 input_selectGO3 <- 'GOBP'  #Gene set category
 input_minSetSize <- 15 #Min gene set size
 input_maxSetSize <- 2000   #Max gene set size 
 GeneSets.out <-readGeneSets( geneSetFile,
    convertedData.out, input_selectGO3,input_selectOrg,
    c(input_minSetSize, input_maxSetSize)  )  
 # Alternatively, users can use their own GMT files by
 #GeneSets.out <- readGMTRobust('somefile.GMT')  
 results <- KmeansGO()  #Enrichment analysis for k-Means clusters   
 results$adj.Pval <- format( results$adj.Pval,digits=3 )
 kable( results, row.names=FALSE) %>%
  kable_styling(bootstrap_options = c("striped", "hover")) %>%
  scroll_box(width = "100%") 

Cluster	adj.Pval	Genes	Pathways
A	2.25e-169	236	Amide biosynthetic process
	7.51e-167	225	Translation
	1.76e-166	225	Peptide biosynthetic process
	2.01e-161	313	Organonitrogen compound biosynthetic process
	2.03e-159	242	Cellular amide metabolic process
	4.21e-158	226	Peptide metabolic process
	1.69e-89	137	Ribonucleoprotein complex biogenesis
	5.02e-83	206	Cellular component biogenesis
	1.94e-71	109	Ribosome biogenesis
	1.93e-62	95	Response to cadmium ion
B	1.41e-14	17	Detoxification
	2.90e-14	19	Response to toxic substance
	2.90e-14	16	Drug catabolic process
	2.90e-14	15	Cellular oxidant detoxification
	3.83e-14	10	Water transport
	3.83e-14	10	Fluid transport
	3.83e-14	15	Cellular detoxification
	4.93e-14	12	Hydrogen peroxide catabolic process
	7.79e-14	15	Cellular response to toxic substance
	2.35e-13	20	Response to oxidative stress
C	1.83e-32	129	Response to abiotic stimulus
	2.31e-31	83	Response to inorganic substance
	4.80e-25	54	Response to metal ion
	1.10e-23	88	Cellular catabolic process
	4.30e-23	96	Catabolic process
	5.18e-23	45	Response to cadmium ion
	5.68e-22	93	Response to oxygen-containing compound
	7.79e-21	87	Oxidation-reduction process
	9.68e-21	88	Cellular response to chemical stimulus
	6.58e-19	74	Response to acid chemical
D	1.66e-16	51	Response to abiotic stimulus
	1.66e-16	20	Cellular response to decreased oxygen levels
	1.66e-16	43	Cellular response to chemical stimulus
	1.66e-16	20	Cellular response to oxygen levels
	1.66e-16	20	Cellular response to hypoxia
	7.26e-16	38	Cellular response to stress
	9.22e-16	20	Response to hypoxia
	1.07e-15	20	Response to decreased oxygen levels
	1.07e-15	20	Response to oxygen levels
	2.58e-15	39	Response to external stimulus

 input_seedTSNE <- 0    #Random seed for t-SNE
 input_colorGenes <- TRUE   #Color genes in t-SNE plot? 
 tSNEgenePlot()  #Plot genes using t-SNE 

5. PCA and beyond

 input_selectFactors <- 'Variety'   #Factor coded by color
 input_selectFactors2 <- 'Light'    #Factor coded by shape
 input_tsneSeed2 <- 0   #Random seed for t-SNE 
 #PCA, MDS and t-SNE plots
 PCAplot()  

 MDSplot() 

 tSNEplot()  

 #Read gene sets for pathway analysis using PGSEA on principal components 
 input_selectGO6 <- 'GOBP' 
 GeneSets.out <-readGeneSets( geneSetFile,
    convertedData.out, input_selectGO6,input_selectOrg,
    c(input_minSetSize, input_maxSetSize)  )  
 PCApathway() # Run PGSEA analysis 

## Warning: Package 'KEGG.db' is deprecated and will be removed from Bioconductor
##   version 3.12

 cat( PCA2factor() )   #The correlation between PCs with factors 

## 
##  Correlation between Principal Components (PCs) with factors
## PC1 is correlated with Light (p=5.73e-03).
## PC2 is correlated with Variety (p=2.38e-06).
## PC5 is correlated with Gravity (p=1.07e-05).

6. DEG1

 input_CountsDEGMethod <- 3 #DESeq2= 3,limma-voom=2,limma-trend=1 
 input_limmaPval <- 0.1 #FDR cutoff
 input_limmaFC <- 2 #Fold-change cutoff
 input_selectModelComprions <- 'Gravity: Microgravity vs. Terrestrial'  #Selected comparisons
 input_selectFactorsModel <- 'Gravity'  #Selected comparisons
 input_selectInteractions <- NULL   #Selected comparisons
 input_selectBlockFactorsModel <- NULL  #Selected comparisons
 factorReferenceLevels.out <- c('Gravity:Terrestrial','Light:Alight') 

 limma.out <- limma()

## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors

 DEG.data.out <- DEG.data()
 limma.out$comparisons 

## [1] "Microgravity-Terrestrial"

 input_selectComparisonsVenn = limma.out$comparisons[1:3] # use first three comparisons
 input_UpDownRegulated <- FALSE #Split up and down regulated genes 
 vennPlot() # Venn diagram 

  sigGeneStats() # number of DEGs as figure 

  sigGeneStatsTable() # number of DEGs as table 

##                                       Comparisons Up Down
## Microgravity-Terrestrial Microgravity-Terrestrial 77   50

7. DEG2

 input_selectContrast <- 'Microgravity-Terrestrial' #Selected comparisons 
 selectedHeatmap.data.out <- selectedHeatmap.data()
 selectedHeatmap()   # heatmap for DEGs in selected comparison

## $genes
##           Col0_GC_Alight_Rep1 Col0_GC_Alight_Rep2 Col0_GC_Alight_Rep3
## AT3G62040            8.104727           11.568320           11.697708
## AT5G05960            7.184797           10.242935           10.254254
## AT5G13930            9.633729            8.863853            9.079192
## AT2G21640            4.220106            5.369623            5.263637
## AT4G31910            8.778767            9.839191           10.017478
## AT3G03780            8.068476            7.514599            7.906864
## AT2G40900            8.348801            9.525778            9.824455
## AT2G41480            6.566055            9.736564            9.515016
## AT5G63850            6.624296            9.016747            9.247347
## AT3G54500            8.967762            8.413979            8.572169
## AT3G02850            7.184797            6.833750            7.084960
## AT5G37260            7.074229            7.992095            8.451734
## AT4G31730            7.094973            8.471031            8.703877
## AT3G23810            6.505365            7.857968            7.333875
## AT3G17609            8.031291            7.545284            7.598275
## AT3G26830            5.582295            6.676309            6.976491
## AT5G05270            7.602012            7.782703            7.717650
## AT2G46830            7.063744            7.213971            7.126162
## AT3G51240            7.895992            7.223490            6.965181
## AT3G09600            7.194441            7.756716            7.796338
## AT5G17300            7.241716            7.814542            8.175731
## AT5G47990            2.000000            2.987502            3.444032
## AT4G01440            2.573281            5.859255            6.369208
## AT4G09950            5.177983            5.225486            5.650070
## AT1G78580            6.624296            6.918234            6.484596
## AT5G50120            5.552608            7.052461            7.491961
## AT3G54820            4.060523            3.981213            4.734235
## AT5G44350            3.973583            5.677282            5.810333
## AT2G47560            2.000000            6.298089            7.185841
## AT3G57020            5.921170            5.531638            5.908068
## AT3G06020            2.000000            6.986908            6.298877
## AT5G08640            5.800066            5.065335            5.784828
## AT3G03770            4.494437            5.262893            4.439601
## AT2G25680            4.142520            3.568275            3.790058
## AT1G30380            4.555580            5.953783            5.758865
## AT3G56730            3.150403            6.185717            6.634906
## AT5G56720            4.060523            4.228145            4.372699
## AT5G64940            6.288384            5.976478            4.885877
## AT2G47460            5.098033            5.784058            5.678047
## AT2G02020            3.676072            3.683106            3.790058
## AT5G50800            2.000000            2.796649            3.156490
## AT4G24670            4.670600            4.372029            4.786574
## AT1G29910            5.360388            6.020824            4.503539
## AT1G11700            2.792319            5.369623            5.931501
## AT4G25830            4.494437            5.147632            5.650070
## AT4G15430            4.494437            4.679226            5.335664
## AT4G26790            3.973583            4.885159            3.568825
## AT1G68585            3.150403            4.438924            4.228798
## AT5G13630            3.973583            5.262893            3.981833
## AT1G62710            2.314931            4.301880            3.981833
## AT3G05880           11.327215           12.203435           11.984178
## AT3G44300           11.440452           11.115770           10.747271
## AT2G26400            5.582295            3.789468            5.023084
## AT3G54040            6.624296            9.213468            9.739051
## AT1G19960            8.318253            9.678921            9.112891
## AT5G23220            8.504212            7.845693            7.777065
## AT5G24530            7.847918            7.906048            8.402275
## AT5G22555            6.988136            7.418421            7.648631
## AT3G56400            7.184797            6.298089            7.009896
## AT5G05500            6.095994            7.975291            8.233434
## AT5G66170            6.872751            7.341832            6.591435
## AT1G56300            8.063223            7.459277            6.796771
## AT1G51420            7.658846            7.763257            7.620072
## AT5G01210            6.156451            5.704726            6.224941
## AT3G21510            6.032891            7.857968            8.406462
## AT5G66780            5.639896            3.443507            2.987914
## AT3G22121            7.323093            4.836365            6.809475
## AT5G07010            8.052657            7.041741            6.822067
## AT5G41280            6.075265            6.545817            6.147010
## AT5G13330            6.680276            6.084888            6.205850
## AT1G49720            7.287490            6.821266            7.289225
## AT3G22120            7.481150            4.564073            5.758865
## AT2G46680            6.095994            6.870564            6.515937
## AT5G15120            6.580837            4.068305            4.564763
## AT3G53160            7.890070            4.502855            4.372699
## AT2G18690            2.000000            4.932356            5.066066
## AT2G15960            4.876320            3.789468            4.439601
## AT3G17790            4.060523            4.885159            5.107804
## AT4G13195            7.063744            4.228145            5.107804
## AT5G02810            7.496823            6.084888            6.316784
## AT4G13390            3.150403            4.836365            5.370373
## AT2G44080            5.459697            5.500658            5.860028
## AT1G12040            3.973583            6.261596            6.106407
## AT1G22770            6.054234            6.368419            6.298877
## AT4G33070            6.442008            5.403514            5.107804
## AT3G52770            5.695286            5.930725            5.784828
## AT4G33980            4.827574            5.591671            5.226228
## AT2G43570            3.300833            5.468998            5.300100
## AT2G36470            4.430589            6.063847            5.732426
## AT1G06460            6.306284            4.502855            5.532397
## AT1G48930            4.614236            5.930725            5.148368
## AT5G48175            4.494437            5.953783            5.931501
## AT5G02230            3.676072            3.789468            4.228798
## AT1G11210            2.573281            5.187084            4.734235
## AT4G12290            5.459697            3.888524            4.228798
## AT5G18470            3.150403            3.789468            3.568825
## AT3G23150            5.253734            4.372029            3.889130
## AT5G57540            2.000000            2.987502            2.987914
## AT5G24470            5.290169            5.147632            4.439601
## AT5G61650            3.782207            3.568275            4.885877
## AT1G02450            2.982442            3.306918            2.987914
## AT5G19790            2.792319            5.976478            4.679927
## AT2G28690            4.777124            4.622798            4.439601
## AT2G05520            2.314931            2.576643            3.790058
## AT2G20520            2.000000            2.000000            3.444032
## AT5G42900            4.430589            4.301880            4.933077
## AT1G54970            2.000000            2.316942            3.156490
## AT5G22270            2.982442            3.789468            2.987914
## AT2G20560            4.555580            4.836365            5.148368
## AT1G28480            2.982442            3.156032            3.307413
## AT3G53800            5.325706            4.978058            4.933077
## AT5G61660            4.142520            3.888524            4.933077
## AT5G18270            3.300833            4.622798            4.885877
## AT3G10040            4.430589            3.981213            3.156490
## AT4G03440            4.555580            5.022356            4.933077
## AT3G04070            2.314931            3.306918            2.987914
## AT3G10710            2.000000            4.502855            3.444032
## AT1G79320            2.000000            3.789468            3.568825
## AT1G64480            2.000000            3.306918            2.987914
## AT3G05930            4.220106            4.068305            5.023084
## AT1G01660            3.300833            4.228145            3.790058
## AT5G25190            2.573281            3.683106            2.797002
## AT4G35180            2.000000            2.000000            2.000000
## AT1G16440            2.000000            3.981213            2.797002
## AT2G46860            2.000000            2.796649            2.576916
## AT1G25240            2.000000            2.316942            2.797002
## AT1G79860            2.314931            2.796649            2.000000
##           Ws_GC_Alight_Rep1 Ws_GC_Alight_Rep2 Ws_GC_Alight_Rep3
## AT3G62040          8.612741          8.041874          7.864678
## AT5G05960          7.580363          9.495186          7.621008
## AT5G13930          5.989479         10.560442          6.642980
## AT2G21640          5.138793          4.372448          4.674539
## AT4G31910          8.686364          8.575583          8.227133
## AT3G03780          5.825356          7.410616          7.834058
## AT2G40900          8.832511          8.150684          9.022141
## AT2G41480          6.324211          6.871066          7.169855
## AT5G63850          8.875117          7.846203          9.134463
## AT3G54500          8.710093          8.130581          8.305114
## AT3G02850          8.318510          8.435128          8.624284
## AT5G37260          7.602267          8.346402          8.357727
## AT4G31730          7.666051          6.717809          7.846384
## AT3G23810          5.611622          8.355091          7.711386
## AT3G17609          7.720473          7.987027          7.870725
## AT3G26830          5.216588          5.784540          3.679283
## AT5G05270          7.074482          8.337660          7.119960
## AT2G46830          7.779341          7.105406          7.649425
## AT3G51240          6.908597          8.068535          6.711965
## AT3G09600          7.213790          6.895098          7.236931
## AT5G17300          7.473503          6.704271          6.853033
## AT5G47990          2.792429          5.403984          2.574815
## AT4G01440          5.800308          4.068700          5.142704
## AT4G09950          6.375989          5.187546          6.525247
## AT1G78580          6.920270          6.546314          6.446465
## AT5G50120          4.969359          4.503283          5.878295
## AT3G54820          2.000000          4.503283          2.574815
## AT5G44350          4.923698          7.507332          2.984751
## AT2G47560          5.013619          4.679664          3.884471
## AT3G57020          4.293939          6.834251          4.927529
## AT3G06020          5.013619          3.156318          2.574815
## AT5G08640          4.876545          7.115670          5.102160
## AT3G03770          4.827798          4.679664          4.831589
## AT2G25680          3.437212          3.789837          3.152972
## AT1G30380          5.668097          5.999308          3.884471
## AT3G56730          3.561676          4.503283          3.679283
## AT5G56720          5.394492          3.156318          5.142704
## AT5G64940          6.270506          5.835105          5.556883
## AT2G47460          5.178215          6.262088          5.556883
## AT2G02020          3.973777          3.307227          2.000000
## AT5G50800          3.150546          2.796870          2.984751
## AT4G24670          4.614454          4.885608          5.644194
## AT1G29910          5.668097          4.372448          3.977066
## AT1G11700          4.614454          4.786308          2.984751
## AT4G25830          2.000000          5.107529          2.000000
## AT4G15430          4.220311          3.888903          5.220526
## AT4G26790          4.614454          3.443835          3.977066
## AT1G68585          2.982571          3.789837          4.223777
## AT5G13630          4.725066          3.888903          2.574815
## AT1G62710          4.827798          3.683464          3.679283
## AT3G05880         10.871893         11.963414         11.914083
## AT3G44300         10.834598         10.480302         11.042870
## AT2G26400          6.033136          5.954268          7.809085
## AT3G54040          6.136824          8.662156          8.109331
## AT1G19960          7.937038          9.779657          8.617107
## AT5G23220          7.727134          8.864367          8.700951
## AT5G24530          6.721130          8.020185          8.453502
## AT5G22555          3.437212          8.644544          7.099508
## AT3G56400          6.033136          7.669381          6.725375
## AT5G05500          2.314982          8.665653          4.674539
## AT5G66170          7.375155          7.242845          7.387917
## AT1G56300          6.195639          6.676808          6.256330
## AT1G51420          6.011473          7.802400          7.731462
## AT5G01210          5.800308          6.605773          6.628778
## AT3G21510          5.695526          7.583254          6.901093
## AT5G66780          4.494651          3.789837          4.618146
## AT3G22121          7.602267          6.846627          7.516897
## AT5G07010          6.908597          6.419498          7.169855
## AT5G41280          6.096239          7.821336          7.997721
## AT5G13330          5.098263          7.020560          7.697845
## AT1G49720          6.233558          6.690605          7.777248
## AT3G22120          7.543101          7.315877          7.846384
## AT2G46680          6.341678          6.744509          6.670971
## AT5G15120          4.494651          5.931210          6.292817
## AT3G53160          6.786364          5.954268          6.642980
## AT2G18690          3.150546          5.107529          2.984751
## AT2G15960          4.363992          5.187546          6.397045
## AT3G17790          3.782391          5.263358          5.182140
## AT4G13195          5.944461          5.469470          6.079660
## AT5G02810          6.721130          5.784540          6.363132
## AT4G13390          2.000000          2.796870          2.794295
## AT2G44080          5.013619          7.185538          5.925534
## AT1G12040          2.314982          5.883958          3.152972
## AT1G22770          6.931848          4.978511          6.100393
## AT4G33070          5.800308          5.501131          5.971275
## AT3G52770          4.876545          5.107529          5.142704
## AT4G33980          3.676251          6.085376          5.854082
## AT2G43570          2.314982          4.932807          3.679283
## AT2G36470          3.150546          5.562442          5.102160
## AT1G06460          4.670820          4.503283          5.672161
## AT1G48930          2.314982          5.677761          3.152972
## AT5G48175          3.676251          5.065792          5.398484
## AT5G02230          2.000000          4.978511          4.064076
## AT1G11210          2.982571          4.786308          3.152972
## AT4G12290          5.552846          4.439348          5.495594
## AT5G18470          3.881255          5.403984          3.439996
## AT3G23150          2.792429          4.228553          5.294364
## AT5G57540          2.000000          2.000000          2.000000
## AT5G24470          5.459934          5.621253          6.446465
## AT5G61650          2.573366          4.439348          3.679283
## AT1G02450          2.573366          3.888903          3.564595
## AT5G19790          2.792429          5.022811          2.984751
## AT2G28690          2.000000          3.789837          2.984751
## AT2G05520          2.314982          2.796870          2.000000
## AT2G20520          2.000000          2.000000          2.000000
## AT5G42900          3.973777          4.150840          4.297452
## AT1G54970          2.000000          2.000000          2.000000
## AT5G22270          2.000000          3.307227          2.574815
## AT2G20560          4.725066          4.302294          4.434395
## AT1G28480          3.300988          4.733971          2.574815
## AT3G53800          3.561676          5.107529          4.297452
## AT5G61660          2.573366          4.372448          4.434395
## AT5G18270          2.792429          4.302294          4.146135
## AT3G10040          3.782391          2.987760          3.679283
## AT4G03440          4.060721          5.022811          4.297452
## AT3G04070          3.676251          2.000000          4.880356
## AT3G10710          2.000000          2.796870          2.000000
## AT1G79320          2.000000          5.065792          2.000000
## AT1G64480          2.573366          4.623233          3.679283
## AT3G05930          3.881255          5.225950          4.064076
## AT1G01660          3.782391          4.439348          4.367548
## AT5G25190          2.314982          2.796870          2.574815
## AT4G35180          3.973777          3.307227          3.439996
## AT1G16440          2.314982          2.000000          2.000000
## AT2G46860          2.000000          2.000000          2.000000
## AT1G25240          2.792429          2.576814          2.000000
## AT1G79860          2.000000          2.000000          2.000000
##           Col0PhyD_GC_Alight_Rep1 Col0PhyD_GC_Alight_Rep2
## AT3G62040                9.330313                7.396435
## AT5G05960                9.144908                6.563949
## AT5G13930                2.000000                9.057714
## AT2G21640                5.299274                3.157781
## AT4G31910                9.255741                8.268367
## AT3G03780                8.678954                6.733772
## AT2G40900                7.917734                8.650708
## AT2G41480                7.935217                6.354170
## AT5G63850                7.084068                6.066828
## AT3G54500                6.730635                9.170510
## AT3G02850                7.251587                7.746656
## AT5G37260                8.297052                7.344924
## AT4G31730                7.155417                6.897659
## AT3G23810                7.633521                7.188116
## AT3G17609                3.306865                7.600566
## AT3G26830                6.952893                4.070720
## AT5G05270                5.022278                6.405358
## AT2G46830                4.885082                7.362299
## AT3G51240                2.000000                6.470901
## AT3G09600                7.223403                7.657977
## AT5G17300                6.676224                7.118244
## AT5G47990                4.502782                3.685290
## AT4G01440                7.675735                4.152894
## AT4G09950                6.757090                7.600566
## AT1G78580                5.591590                4.441510
## AT5G50120                6.368334                5.406385
## AT3G54820                5.369542                3.570377
## AT5G44350                5.225407                6.679355
## AT2G47560                2.000000                2.317568
## AT3G57020                3.306865                6.693153
## AT3G06020                4.563999                2.317568
## AT5G08640                2.000000                4.566707
## AT3G03770                4.836289                4.887900
## AT2G25680                2.796612                3.570377
## AT1G30380                3.443450                6.720359
## AT3G56730                4.932279                3.983581
## AT5G56720                5.369542                6.354170
## AT5G64940                2.796612                5.837570
## AT2G47460                2.000000                5.956748
## AT2G02020                5.907210                5.025136
## AT5G50800                2.000000                2.000000
## AT4G24670                4.502782                4.980826
## AT1G29910                3.683045                5.439505
## AT1G11700                4.733454                4.304407
## AT4G25830                2.000000                4.980826
## AT4G15430                3.981146                5.265733
## AT4G26790                4.438852                4.505466
## AT1G68585                3.306865                4.625454
## AT5G13630                2.000000                4.681902
## AT1G62710                4.502782                4.736224
## AT3G05880               11.302960               12.551491
## AT3G44300                9.699441               10.824388
## AT2G26400                7.483160                7.001113
## AT3G54040                7.393254                9.594727
## AT1G19960                7.851756               10.250767
## AT5G23220                7.393254                8.845534
## AT5G24530                7.073581                9.431463
## AT5G22555                3.306865                8.978238
## AT3G56400                7.145437                9.398155
## AT5G05500                4.371957                8.792043
## AT5G66170                7.736826                8.295756
## AT1G56300                6.402253                8.357710
## AT1G51420                6.833664                8.555726
## AT5G01210                6.952893                7.012163
## AT3G21510                6.530477                8.211979
## AT5G66780                3.306865                4.625454
## AT3G22121                5.187005                7.138555
## AT5G07010                7.084068                8.231021
## AT5G41280                5.731575                7.486346
## AT5G13330                7.125269                7.798636
## AT1G49720                7.213884                7.370909
## AT3G22120                6.204980                7.327336
## AT2G46680                6.619679                7.087231
## AT5G15120                7.801803                7.772880
## AT3G53160                6.929823                7.107980
## AT2G18690                5.334836                7.867182
## AT2G15960                6.385393                6.246000
## AT3G17790                5.147553                6.087871
## AT4G13195                6.648228                7.273250
## AT5G02810                5.859172                8.039093
## AT4G13390                4.150368                6.989978
## AT2G44080                6.605190                7.370909
## AT1G12040                4.885082                6.978756
## AT1G22770                5.561885                7.023129
## AT4G33070                5.731575                6.188713
## AT3G52770                4.371957                6.129058
## AT4G33980                4.885082                6.578890
## AT2G43570                4.228075                5.761032
## AT2G36470                5.299274                6.747061
## AT1G06460                5.262813                6.371435
## AT1G48930                3.683045                6.336696
## AT5G48175                4.977980                6.354170
## AT5G02230                3.155983                5.189907
## AT1G11210                3.683045                6.246000
## AT4G12290                4.371957                6.169101
## AT5G18470                3.306865                4.625454
## AT3G23150                6.105541                6.188713
## AT5G57540                2.796612                3.890838
## AT5G24470                4.679151                4.566707
## AT5G61650                3.306865                5.837570
## AT1G02450                2.316924                2.797995
## AT5G19790                2.316924                5.302202
## AT2G28690                3.443450                4.788575
## AT2G05520                5.299274                4.230639
## AT2G20520                2.316924                2.000000
## AT5G42900                4.563999                5.503548
## AT1G54970                2.000000                2.000000
## AT5G22270                4.977980                4.304407
## AT2G20560                4.228075                5.707653
## AT1G28480                2.000000                6.608315
## AT3G53800                5.591590                5.109873
## AT5G61660                2.000000                5.439505
## AT5G18270                4.885082                5.228318
## AT3G10040                6.166023                3.791722
## AT4G03440                4.679151                4.230639
## AT3G04070                4.622723                3.791722
## AT3G10710                2.316924                4.304407
## AT1G79320                2.796612                3.308808
## AT1G64480                2.000000                2.577688
## AT3G05930                3.981146                4.304407
## AT1G01660                3.443450                3.308808
## AT5G25190                3.981146                2.000000
## AT4G35180                2.000000                2.000000
## AT1G16440                2.796612                2.797995
## AT2G46860                2.796612                2.317568
## AT1G25240                2.796612                2.000000
## AT1G79860                2.000000                2.000000
##           Col0PhyD_GC_Alight_Rep3 Col0_FLT_Alight_Rep1 Col0_FLT_Alight_Rep2
## AT3G62040                9.894806             7.136576             6.996681
## AT5G05960               10.000006             4.486833             5.797668
## AT5G13930                9.251930             2.000000             2.314424
## AT2G21640                4.507339             4.135370             3.971658
## AT4G31910                8.946281             8.566906             8.713985
## AT3G03780                6.899856             6.827330             7.029320
## AT2G40900                9.425467             6.315177             7.112914
## AT2G41480                6.595856             3.874333             4.966879
## AT5G63850                8.595251             7.415879             7.229869
## AT3G54500                9.008396             6.297503             6.650077
## AT3G02850                8.983308             5.935570             7.040038
## AT5G37260                8.595251             4.915445             5.692902
## AT4G31730                8.406834             5.740067             5.772181
## AT3G23810                6.911725             6.243141             6.390135
## AT3G17609                8.209421             3.295345             3.559797
## AT3G26830                6.025932             4.486833             4.492313
## AT5G05270                7.302762             4.769227             5.095752
## AT2G46830                8.046723             3.295345             2.791227
## AT3G51240                7.256974             2.977881             2.314424
## AT3G09600                7.905477             3.966694             4.612075
## AT5G17300                7.519923             4.356334             3.879184
## AT5G47990                4.841048             2.000000             3.435419
## AT4G01440                5.536611             3.874333             5.918758
## AT4G09950                6.722546             5.352042             5.871534
## AT1G78580                6.722546             6.224555             5.822713
## AT5G50120                6.923497             4.769227             4.612075
## AT3G54820                2.990203             2.000000             4.291676
## AT5G44350                5.958875             3.145328             4.140522
## AT2G47560                6.303256             2.313121             2.314424
## AT3G57020                6.131201             4.356334             2.791227
## AT3G06020                4.982811             2.000000             2.000000
## AT5G08640                5.191931             2.788417             2.000000
## AT3G03770                4.790519             3.775652             4.722653
## AT2G25680                3.985279             2.977881             4.058559
## AT1G30380                6.248153             2.570255             2.981167
## AT3G56730                5.981575             2.788417             2.791227
## AT5G56720                3.793339             3.295345             3.879184
## AT5G64940                5.682300             3.145328             4.361702
## AT2G47460                6.151363             2.570255             2.791227
## AT2G02020                5.958875             4.212847             3.879184
## AT5G50800                4.376422             2.000000             2.000000
## AT4G24670                5.374540             4.547908             4.722653
## AT1G29910                4.507339             2.000000             3.435419
## AT1G11700                5.596664             2.788417             3.435419
## AT4G25830                5.267770             2.570255             2.572435
## AT4G15430                4.841048             3.431221             3.559797
## AT4G26790                4.072452             3.669723             2.572435
## AT1G68585                5.441569             2.570255             2.791227
## AT5G13630                5.152462             2.313121             2.572435
## AT1G62710                3.892499             2.570255             3.435419
## AT3G05880               12.235679            11.412351            11.785893
## AT3G44300               10.474249            12.045572            11.398331
## AT2G26400                5.596664             8.850617             9.217365
## AT3G54040                9.593308             6.671388             8.397157
## AT1G19960                9.439883             8.427604             8.848106
## AT5G23220                7.715420            10.195167            10.163114
## AT5G24530                8.937600             8.514319             8.599096
## AT5G22555                7.980648             8.606921             8.602715
## AT3G56400                7.715420             7.750650             8.205766
## AT5G05500                6.875822             7.649780             8.627799
## AT5G66170                6.667608             8.369577             7.355317
## AT1G56300                6.839002             8.610504             8.266352
## AT1G51420                7.456517             8.798163             8.454365
## AT5G01210                7.869416             6.956833             8.350561
## AT3G21510                7.881537             7.456265             7.724344
## AT5G66780                2.990203             5.602892             5.391919
## AT3G22121                7.646065             7.571052             8.388796
## AT5G07010                7.311747             8.621200             8.132543
## AT5G41280                6.958247             8.378011             7.690730
## AT5G13330                6.639334             7.763582             7.795649
## AT1G49720                7.440220             8.431661             8.652455
## AT3G22120                7.320677             7.348813             7.494297
## AT2G46680                6.489003             6.279609             6.321510
## AT5G15120                5.111882             6.511933             6.373283
## AT3G53160                5.152462             7.464208             7.509801
## AT2G18690                5.839684             6.685046             5.895339
## AT2G15960                5.267770             6.066565             5.425005
## AT3G17790                5.111882             5.791482             5.550245
## AT4G13195                6.722546             7.832706             7.968307
## AT5G02810                7.130643             7.391093             7.826930
## AT4G13390                3.159035             5.544149             7.462780
## AT2G44080                5.339821             7.022859             6.846030
## AT1G12040                4.507339             6.024208             7.248472
## AT1G22770                5.912374             7.731032             7.776549
## AT4G33070                6.068966             7.251192             6.882218
## AT3G52770                5.536611             5.208104             4.553436
## AT4G33980                5.935812             5.958246             6.093562
## AT2G43570                5.709752             4.769227             4.612075
## AT2G36470                6.303256             5.317381             6.303831
## AT1G06460                5.789107             6.629618             6.321510
## AT1G48930                4.790519             4.769227             6.212033
## AT5G48175                5.566950             6.107714             6.192951
## AT5G02230                4.683821             4.769227             4.361702
## AT1G11210                4.841048             5.245457             3.674297
## AT4G12290                5.596664             6.465202             6.321510
## AT5G18470                3.446951             5.317381             6.249456
## AT3G23150                4.376422             6.814960             6.963285
## AT5G57540                2.000000             2.000000             5.550245
## AT5G24470                5.763137             6.433185             6.548647
## AT5G61650                4.306221             4.053496             5.847330
## AT1G02450                3.571885             3.669723             4.058559
## AT5G19790                3.686857             4.212847             6.212033
## AT2G28690                4.232428             5.451291             5.692902
## AT2G05520                3.892499             4.053496             4.140522
## AT2G20520                2.000000             2.000000             5.941802
## AT5G42900                5.152462             5.602892             5.214051
## AT1G54970                2.000000             2.788417             5.579927
## AT5G22270                2.990203             3.555395             4.428485
## AT2G20560                4.376422             3.775652             4.361702
## AT1G28480                2.578438             2.570255             2.572435
## AT3G53800                5.191931             6.433185             6.051806
## AT5G61660                4.232428             3.775652             4.966879
## AT5G18270                5.736692             4.053496             5.579927
## AT3G10040                4.738156             4.868333             5.519940
## AT4G03440                5.267770             5.865324             5.609011
## AT3G04070                3.446951             4.486833             4.428485
## AT3G10710                2.318017             2.788417             6.249456
## AT1G79320                4.443365             4.716998             3.559797
## AT1G64480                3.793339             2.000000             2.981167
## AT3G05930                3.793339             4.053496             3.780375
## AT1G01660                2.990203             4.053496             3.148984
## AT5G25190                2.000000             4.135370             3.559797
## AT4G35180                2.000000             2.977881             3.559797
## AT1G16440                2.990203             2.977881             4.966879
## AT2G46860                2.000000             2.313121             2.572435
## AT1G25240                2.000000             2.000000             3.879184
## AT1G79860                2.000000             2.000000             2.981167
##           Col0_FLT_Alight_Rep3 Ws_FLT_Alight_Rep1 Ws_FLT_Alight_Rep2
## AT3G62040             5.137538           6.196179           7.756117
## AT5G05960             6.883597           6.342221           7.307486
## AT5G13930             2.000000           2.000000           2.000000
## AT2G21640             4.723864           5.098768           5.039530
## AT4G31910             7.154349           7.166122           6.901539
## AT3G03780             7.725745           7.233193           6.971920
## AT2G40900             8.113780           6.596266           7.971832
## AT2G41480             4.875322           5.057064           6.708921
## AT5G63850             6.719768           6.748128           8.256631
## AT3G54500             6.094906           6.681075           7.134173
## AT3G02850             7.259075           5.749396           6.223578
## AT5G37260             6.115340           6.215264           6.144523
## AT4G31730             6.155361           6.157236           7.715668
## AT3G23810             6.408154           5.553369           5.124381
## AT3G17609             3.781387           5.178723           5.609626
## AT3G26830             5.252700           2.000000           4.243559
## AT5G05270             5.773505           4.364452           6.223578
## AT2G46830             4.292813           5.139299           3.995850
## AT3G51240             3.560741           2.982853           2.000000
## AT3G09600             5.215325           4.969858           3.455911
## AT5G17300             4.493487           4.143163           3.166852
## AT5G47990             2.981872           2.000000           2.000000
## AT4G01440             4.219199           3.150860           4.695757
## AT4G09950             3.436320           4.924194           3.803403
## AT1G78580             3.880224           4.143163           5.972096
## AT5G50120             3.781387           3.881671           4.995157
## AT3G54820             2.791830           3.437572           3.995850
## AT5G44350             5.798994           3.881671           3.166852
## AT2G47560             2.314704           2.573554           2.583119
## AT3G57020             2.791830           3.150860           2.000000
## AT3G06020             2.314704           2.315094           2.000000
## AT5G08640             2.000000           2.000000           2.583119
## AT3G03770             4.429647           4.061155           3.803403
## AT2G25680             3.300147           3.150860           3.581274
## AT1G30380             3.560741           3.676643           3.455911
## AT3G56730             3.675278           2.000000           3.455911
## AT5G56720             3.300147           2.982853           2.583119
## AT5G64940             2.572902           4.220759           2.000000
## AT2G47460             3.300147           2.315094           3.581274
## AT2G02020             2.791830           2.000000           2.000000
## AT5G50800             2.000000           2.000000           3.581274
## AT4G24670             3.436320           3.881671           2.583119
## AT1G29910             2.981872           3.676643           3.318609
## AT1G11700             2.791830           2.982853           5.082579
## AT4G25830             2.572902           2.000000           2.583119
## AT4G15430             3.436320           2.000000           2.320823
## AT4G26790             3.300147           2.792670           2.997235
## AT1G68585             3.436320           2.315094           4.243559
## AT5G13630             2.572902           2.573554           2.000000
## AT1G62710             2.572902           3.301327           3.995850
## AT3G05880            12.943849          12.092880          13.852058
## AT3G44300            11.511421          11.963066          10.585065
## AT2G26400             8.780824          10.331928           9.719004
## AT3G54040            10.173091           8.185137          11.521948
## AT1G19960            10.193656           9.379332          11.221487
## AT5G23220            10.069555          10.415780           9.447766
## AT5G24530            10.132313           8.620482           9.545240
## AT5G22555             8.946798           9.707130          10.199054
## AT3G56400             9.864534           7.971667           8.256631
## AT5G05500             8.909256           9.383556           9.964592
## AT5G66170             9.048593           8.641798           9.184326
## AT1G56300             8.994463           7.971667           7.814741
## AT1G51420             8.784019           8.375301           7.251997
## AT5G01210             8.891594           7.551189           8.756066
## AT3G21510             8.173465           8.524291           9.358632
## AT5G66780             4.292813           5.722971           3.318609
## AT3G22121             6.975885           8.740605           9.194174
## AT5G07010             9.156058           7.010951           6.437662
## AT5G41280             7.952779           9.128135           8.429156
## AT5G13330             8.381797           8.287864           7.858745
## AT1G49720             8.712049           8.175425           8.408157
## AT3G22120             7.657714           8.524291           8.840002
## AT2G46680             9.246838           6.324754           7.701929
## AT5G15120             7.339442           5.395009           8.742708
## AT3G53160             7.924108           6.909149           6.533861
## AT2G18690             8.299361           7.350040           5.387309
## AT2G15960             7.083517           6.734965           7.988904
## AT3G17790             7.114300           5.217098           8.886294
## AT4G13195             6.733053           6.812204           6.609400
## AT5G02810             7.952779           7.588260           7.695010
## AT4G13390             5.920091           5.553369           6.594605
## AT2G44080             7.313147           7.489820           8.270660
## AT1G12040             6.964667           6.920822           7.412446
## AT1G22770             7.894856           6.490579           7.039026
## AT4G33070             7.083517           7.126347           7.971832
## AT3G52770             5.097013           5.139299           6.387045
## AT4G33980             7.705670           6.521573           7.960337
## AT2G43570             7.083517           4.143163           6.352286
## AT2G36470             7.144441           5.460453           7.103149
## AT1G06460             6.759260           5.800838           5.949018
## AT1G48930             6.679165           6.474828           7.478404
## AT5G48175             6.930479           6.458904           6.749557
## AT5G02230             6.594362           4.924194           4.243559
## AT1G11210             6.930479           2.792670           6.280150
## AT4G12290             6.733053           5.967680           5.827743
## AT5G18470             5.747557           5.428102           6.223578
## AT3G23150             6.975885           5.944995           6.695117
## AT5G57540             3.972721           4.828289           4.580350
## AT5G24470             6.269163           6.289172           5.352557
## AT5G61650             5.638833           5.553369           4.949376
## AT1G02450             4.776137           6.176839           5.242975
## AT5G19790             4.723864           5.850508           6.404116
## AT2G28690             6.608846           5.696053           6.060884
## AT2G05520             4.669626           2.000000           2.000000
## AT2G20520             4.362852           2.000000           2.000000
## AT5G42900             6.975885           5.696053           5.877483
## AT1G54970             4.292813           4.431266           2.000000
## AT5G22270             5.521243           4.431266           6.387045
## AT2G20560             5.359345           4.220759           6.827548
## AT1G28480             6.472931           4.364452           3.166852
## AT3G53800             5.012379           5.553369           5.609626
## AT5G61660             5.943137           5.057064           4.902094
## AT5G18270             5.824040           5.850508           5.667324
## AT3G10040             5.176956           4.969858           6.652895
## AT4G03440             5.798994           6.033672           5.387309
## AT3G04070             5.773505           4.364452           6.082253
## AT3G10710             5.426300           3.676643           4.387837
## AT1G79320             4.219199           5.326454           5.486810
## AT1G64480             3.675278           3.782796           5.802215
## AT3G05930             5.012379           4.614932           5.609626
## AT1G01660             5.176956           4.924194           5.722803
## AT5G25190             3.300147           3.974202           5.124381
## AT4G35180             2.000000           4.828289           3.318609
## AT1G16440             3.560741           4.431266           4.083231
## AT2G46860             2.314704           4.061155           3.166852
## AT1G25240             3.149768           3.437572           2.320823
## AT1G79860             3.972721           2.982853           2.320823
##           Ws_FLT_Alight_Rep3 Col0PhyD_FLT_Alight_Rep1 Col0PhyD_FLT_Alight_Rep2
## AT3G62040           7.031744                 6.163084                 6.702935
## AT5G05960           7.053267                 8.740140                 6.881790
## AT5G13930           2.000000                 2.000000                 2.000000
## AT2G21640           4.933077                 4.833612                 4.563350
## AT4G31910           6.809475                 7.448069                 7.008249
## AT3G03780           7.270970                 7.439945                 7.349696
## AT2G40900           7.435714                 7.579610                 7.417571
## AT2G41480           6.205850                 5.104240                 5.833813
## AT5G63850           7.351355                 8.338362                 6.986065
## AT3G54500           6.634907                 5.927733                 7.030096
## AT3G02850           6.224941                 7.398623                 8.007849
## AT5G37260           6.531356                 6.527499                 5.929912
## AT4G31730           6.834551                 6.258561                 6.401509
## AT3G23810           5.860028                 5.588735                 5.975663
## AT3G17609           4.885877                 4.676525                 4.438215
## AT3G26830           2.000000                 4.369446                 3.155553
## AT5G05270           4.734235                 5.400615                 4.149763
## AT2G46830           3.307413                 4.147957                 4.502139
## AT3G51240           2.987914                 2.985916                 3.887889
## AT3G09600           4.734235                 4.299328                 5.368837
## AT5G17300           5.226228                 5.296478                 4.732791
## AT5G47990           2.576916                 2.000000                 2.576356
## AT4G01440           3.568825                 5.856274                 3.155553
## AT4G09950           5.107804                 5.646359                 4.227460
## AT1G78580           2.797002                 5.433722                 4.884407
## AT5G50120           2.987914                 4.500219                 2.576356
## AT3G54820           4.503539                 3.886187                 2.796280
## AT5G44350           4.623494                 3.978823                 3.306400
## AT2G47560           3.156490                 2.575589                 2.576356
## AT3G57020           3.981833                 2.575589                 2.987071
## AT3G06020           2.000000                 2.316311                 2.000000
## AT5G08640           2.000000                 2.316311                 2.000000
## AT3G03770           4.228798                 4.369446                 3.980563
## AT2G25680           2.987914                 3.305011                 2.796280
## AT1G30380           4.151081                 3.566154                 3.442957
## AT3G56730           3.568825                 3.154268                 4.732791
## AT5G56720           3.981833                 5.332032                 2.987071
## AT5G64940           4.439602                 4.436314                 3.980563
## AT2G47460           3.307413                 3.566154                 2.576356
## AT2G02020           3.156490                 2.985916                 3.442957
## AT5G50800           3.156490                 2.000000                 2.316770
## AT4G24670           2.797002                 3.441483                 3.442957
## AT1G29910           3.568825                 2.316311                 2.576356
## AT1G11700           3.683678                 3.978823                 3.682507
## AT4G25830           2.576916                 2.316311                 2.987071
## AT4G15430           2.317106                 3.566154                 2.987071
## AT4G26790           3.790058                 3.305011                 4.067642
## AT1G68585           3.683678                 2.985916                 2.316770
## AT5G13630           2.317106                 2.316311                 2.796280
## AT1G62710           3.307413                 3.441483                 2.796280
## AT3G05880          12.888441                12.673667                13.054522
## AT3G44300          11.557900                11.561850                11.952405
## AT2G26400          10.168102                10.457603                 9.926873
## AT3G54040          10.376678                 9.790403                10.894433
## AT1G19960          10.373473                10.728316                10.674528
## AT5G23220          10.065928                 9.905501                 9.557245
## AT5G24530           8.918439                 9.679190                10.408730
## AT5G22555           9.912464                 9.178902                 7.781848
## AT3G56400           8.530575                 9.254966                 9.284803
## AT5G05500           9.597902                 7.885058                 7.963123
## AT5G66170           8.897769                 8.360052                 9.742330
## AT1G56300           7.523144                 8.848454                 9.219751
## AT1G51420           8.195223                 9.016304                 9.233940
## AT5G01210           7.185841                 9.479646                 9.318483
## AT3G21510           8.998282                 7.943693                 8.077695
## AT5G66780           5.300100                 4.882391                 5.499865
## AT3G22121           8.590686                 7.879061                 8.869064
## AT5G07010           7.289225                 8.275836                 8.960074
## AT5G41280           9.221440                 8.229455                 7.505974
## AT5G13330           8.219223                 8.385657                 8.482108
## AT1G49720           8.252166                 8.439622                 9.013144
## AT3G22120           8.619827                 7.760114                 8.536545
## AT2G46680           6.998847                 8.215245                 8.174054
## AT5G15120           7.243148                 8.252832                 7.417571
## AT3G53160           6.419795                 7.448069                 7.794668
## AT2G18690           6.452559                 6.805586                 7.498157
## AT2G15960           6.998847                 5.927733                 7.940229
## AT3G17790           7.777065                 7.511467                 7.632756
## AT4G13195           6.334472                 7.623314                 7.768914
## AT5G02810           7.402560                 7.848697                 8.441927
## AT4G13390           5.784828                 5.466086                 7.574479
## AT2G44080           7.648631                 7.439945                 7.762402
## AT1G12040           6.531356                 5.400615                 6.756338
## AT1G22770           7.156310                 8.038210                 7.788273
## AT4G33070           7.270970                 7.836371                 7.409260
## AT3G52770           6.021603                 4.833612                 5.648505
## AT4G33980           7.053267                 7.142413                 7.366967
## AT2G43570           4.623494                 6.512082                 6.544984
## AT2G36470           5.908068                 7.112056                 7.857113
## AT1G06460           4.885877                 6.542753                 7.425836
## AT1G48930           6.871366                 5.806588                 5.906481
## AT5G48175           6.186502                 7.049356                 7.278470
## AT5G02230           5.226228                 4.783127                 5.402726
## AT1G11210           5.263637                 6.572785                 7.314523
## AT4G12290           6.452559                 6.415951                 7.030096
## AT5G18470           5.335664                 4.369446                 6.589805
## AT3G23150           6.186502                 6.727640                 6.702935
## AT5G57540           4.734235                 3.566154                 5.619977
## AT5G24470           6.846927                 6.915138                 6.529729
## AT5G61650           5.884248                 5.296478                 7.072823
## AT1G02450           5.437381                 5.701770                 5.648505
## AT5G19790           5.678047                 3.886187                 6.084069
## AT2G28690           5.784828                 6.645240                 4.931600
## AT2G05520           2.317106                 6.330640                 6.450937
## AT2G20520           2.000000                 2.000000                 5.676480
## AT5G42900           6.546612                 5.400615                 5.435838
## AT1G54970           4.503539                 3.305011                 5.676480
## AT5G22270           5.592433                 5.433722                 6.845286
## AT2G20560           5.066066                 5.184234                 5.975663
## AT1G28480           3.981833                 2.575589                 4.678492
## AT3G53800           6.419795                 5.950788                 6.104806
## AT5G61660           5.370373                 4.882391                 6.647478
## AT5G18270           5.621539                 5.466086                 5.676480
## AT3G10040           5.592433                 6.039489                 6.063029
## AT4G03440           6.262383                 5.995820                 5.298572
## AT3G04070           4.623494                 4.369446                 5.703923
## AT3G10710           3.307413                 3.886187                 4.149763
## AT1G79320           3.790058                 3.680902                 3.442957
## AT1G64480           5.226228                 4.225627                 5.368837
## AT3G05930           4.933077                 5.260025                 4.371327
## AT1G01660           4.068937                 4.147957                 3.567698
## AT5G25190           4.734235                 5.019549                 4.622069
## AT4G35180           4.734235                 3.154268                 3.980563
## AT1G16440           2.987914                 3.154268                 3.682507
## AT2G46860           3.981833                 2.575589                 2.000000
## AT1G25240           2.000000                 2.000000                 4.502139
## AT1G79860           3.790058                 2.316311                 2.576356
##           Col0PhyD_FLT_Alight_Rep3
## AT3G62040                 6.510881
## AT5G05960                 6.699485
## AT5G13930                 2.000000
## AT2G21640                 3.977886
## AT4G31910                 8.160672
## AT3G03780                 6.878324
## AT2G40900                 6.346916
## AT2G41480                 6.398092
## AT5G63850                 7.293113
## AT3G54500                 6.080693
## AT3G02850                 5.830478
## AT5G37260                 6.142022
## AT4G31730                 5.221506
## AT3G23810                 6.431227
## AT3G17609                 3.565322
## AT3G26830                 4.224640
## AT5G05270                 4.832533
## AT2G46830                 2.575176
## AT3G51240                 2.985295
## AT3G09600                 4.619065
## AT5G17300                 4.974167
## AT5G47990                 2.000000
## AT4G01440                 4.298327
## AT4G09950                 6.541551
## AT1G78580                 4.619065
## AT5G50120                 4.146985
## AT3G54820                 2.000000
## AT5G44350                 4.146985
## AT2G47560                 2.316064
## AT3G57020                 2.575176
## AT3G06020                 2.000000
## AT5G08640                 2.000000
## AT3G03770                 4.560370
## AT2G25680                 2.794760
## AT1G30380                 4.064912
## AT3G56730                 3.153577
## AT5G56720                 4.435290
## AT5G64940                 2.316064
## AT2G47460                 2.316064
## AT2G02020                 2.575176
## AT5G50800                 2.000000
## AT4G24670                 4.499185
## AT1G29910                 3.153577
## AT1G11700                 2.575176
## AT4G25830                 2.316064
## AT4G15430                 3.153577
## AT4G26790                 3.977886
## AT1G68585                 2.575176
## AT5G13630                 2.794760
## AT1G62710                 2.794760
## AT3G05880                12.809855
## AT3G44300                12.211254
## AT2G26400                 9.020512
## AT3G54040                 9.887934
## AT1G19960                10.129153
## AT5G23220                 9.779536
## AT5G24530                 9.946405
## AT5G22555                 9.143073
## AT3G56400                 9.438213
## AT5G05500                 8.660726
## AT5G66170                 9.750294
## AT1G56300                 9.039439
## AT1G51420                 8.947955
## AT5G01210                 7.636363
## AT3G21510                 9.127994
## AT5G66780                 4.729744
## AT3G22121                 8.228217
## AT5G07010                 8.237613
## AT5G41280                 7.998802
## AT5G13330                 9.031358
## AT1G49720                 8.705428
## AT3G22120                 8.063633
## AT2G46680                 7.058779
## AT5G15120                 8.185160
## AT3G53160                 7.930920
## AT2G18690                 7.692104
## AT2G15960                 8.712184
## AT3G17790                 7.284072
## AT4G13195                 7.470944
## AT5G02810                 8.068906
## AT4G13390                 5.464944
## AT2G44080                 7.161081
## AT1G12040                 7.346197
## AT1G22770                 7.877827
## AT4G33070                 6.495298
## AT3G52770                 5.527567
## AT4G33980                 7.841313
## AT2G43570                 6.161898
## AT2G36470                 7.865758
## AT1G06460                 7.901665
## AT1G48930                 6.541551
## AT5G48175                 6.925598
## AT5G02230                 5.994643
## AT1G11210                 7.363467
## AT4G12290                 6.960063
## AT5G18470                 7.685253
## AT3G23150                 7.110837
## AT5G57540                 2.000000
## AT5G24470                 5.496595
## AT5G61650                 4.298327
## AT1G02450                 4.499185
## AT5G19790                 5.018448
## AT2G28690                 6.526298
## AT2G05520                 6.080693
## AT2G20520                 2.000000
## AT5G42900                 5.805423
## AT1G54970                 2.794760
## AT5G22270                 5.399479
## AT2G20560                 6.902155
## AT1G28480                 3.304264
## AT3G53800                 6.238776
## AT5G61660                 3.786301
## AT5G18270                 4.928483
## AT3G10040                 5.221506
## AT4G03440                 6.059657
## AT3G04070                 5.830478
## AT3G10710                 4.560370
## AT1G79320                 4.064912
## AT1G64480                 5.143678
## AT3G05930                 5.903133
## AT1G01660                 3.786301
## AT5G25190                 5.753969
## AT4G35180                 4.224640
## AT1G16440                 3.565322
## AT2G46860                 2.575176
## AT1G25240                 2.316064
## AT1G79860                 2.985295
## 
## $bar
## AT3G62040 AT5G05960 AT5G13930 AT2G21640 AT4G31910 AT3G03780 AT2G40900 AT2G41480 
##        -1        -1        -1        -1        -1        -1        -1        -1 
## AT5G63850 AT3G54500 AT3G02850 AT5G37260 AT4G31730 AT3G23810 AT3G17609 AT3G26830 
##        -1        -1        -1        -1        -1        -1        -1        -1 
## AT5G05270 AT2G46830 AT3G51240 AT3G09600 AT5G17300 AT5G47990 AT4G01440 AT4G09950 
##        -1        -1        -1        -1        -1        -1        -1        -1 
## AT1G78580 AT5G50120 AT3G54820 AT5G44350 AT2G47560 AT3G57020 AT3G06020 AT5G08640 
##        -1        -1        -1        -1        -1        -1        -1        -1 
## AT3G03770 AT2G25680 AT1G30380 AT3G56730 AT5G56720 AT5G64940 AT2G47460 AT2G02020 
##        -1        -1        -1        -1        -1        -1        -1        -1 
## AT5G50800 AT4G24670 AT1G29910 AT1G11700 AT4G25830 AT4G15430 AT4G26790 AT1G68585 
##        -1        -1        -1        -1        -1        -1        -1        -1 
## AT5G13630 AT1G62710 AT3G05880 AT3G44300 AT2G26400 AT3G54040 AT1G19960 AT5G23220 
##        -1        -1         1         1         1         1         1         1 
## AT5G24530 AT5G22555 AT3G56400 AT5G05500 AT5G66170 AT1G56300 AT1G51420 AT5G01210 
##         1         1         1         1         1         1         1         1 
## AT3G21510 AT5G66780 AT3G22121 AT5G07010 AT5G41280 AT5G13330 AT1G49720 AT3G22120 
##         1         1         1         1         1         1         1         1 
## AT2G46680 AT5G15120 AT3G53160 AT2G18690 AT2G15960 AT3G17790 AT4G13195 AT5G02810 
##         1         1         1         1         1         1         1         1 
## AT4G13390 AT2G44080 AT1G12040 AT1G22770 AT4G33070 AT3G52770 AT4G33980 AT2G43570 
##         1         1         1         1         1         1         1         1 
## AT2G36470 AT1G06460 AT1G48930 AT5G48175 AT5G02230 AT1G11210 AT4G12290 AT5G18470 
##         1         1         1         1         1         1         1         1 
## AT3G23150 AT5G57540 AT5G24470 AT5G61650 AT1G02450 AT5G19790 AT2G28690 AT2G05520 
##         1         1         1         1         1         1         1         1 
## AT2G20520 AT5G42900 AT1G54970 AT5G22270 AT2G20560 AT1G28480 AT3G53800 AT5G61660 
##         1         1         1         1         1         1         1         1 
## AT5G18270 AT3G10040 AT4G03440 AT3G04070 AT3G10710 AT1G79320 AT1G64480 AT3G05930 
##         1         1         1         1         1         1         1         1 
## AT1G01660 AT5G25190 AT4G35180 AT1G16440 AT2G46860 AT1G25240 AT1G79860 
##         1         1         1         1         1         1         1

 # Save gene lists and data into files
 write.csv( selectedHeatmap.data()$genes, 'heatmap.data.csv') 
 write.csv(DEG.data(),'DEG.data.csv' )
 write(AllGeneListsGMT() ,'AllGeneListsGMT.gmt')

 input_selectGO2 <- 'GOBP'  #Gene set category 
 geneListData.out <- geneListData()  
 volcanoPlot()  

  scatterPlot()  

  MAplot()  

  geneListGOTable.out <- geneListGOTable()  
 # Read pathway data again 
 GeneSets.out <-readGeneSets( geneSetFile,
    convertedData.out, input_selectGO2,input_selectOrg,
    c(input_minSetSize, input_maxSetSize)  ) 
 input_removeRedudantSets <- TRUE   #Remove highly redundant gene sets? 
 results <- geneListGO()  #Enrichment analysis
 results$adj.Pval <- format( results$adj.Pval,digits=3 )
 kable( results, row.names=FALSE) %>%
  kable_styling(bootstrap_options = c("striped", "hover")) %>%
  scroll_box(width = "100%") 

Direction	adj.Pval	nGenes	Pathways
Down regulated	3.2e-06	6	Flavonoid metabolic process
	1.4e-05	16	Response to abiotic stimulus
	1.4e-05	5	Flavonoid biosynthetic process
	1.6e-05	6	Circadian rhythm
	1.6e-05	8	Response to auxin
	1.9e-05	6	Rhythmic process
	1.5e-04	9	Response to light stimulus
	1.8e-04	9	Response to radiation
	5.3e-04	3	Neutral amino acid transport
	5.8e-04	4	Response to UV-B
Up regulated	2.0e-06	9	Phosphorelay signal transduction system
	5.4e-06	20	Response to organic substance
	7.0e-06	18	Response to endogenous stimulus
	7.0e-06	18	Response to hormone
	1.7e-05	17	Response to oxygen-containing compound
	2.0e-05	9	Response to organic cyclic compound
	8.9e-05	8	Response to antibiotic
	1.4e-04	16	System development
	1.6e-04	10	Intracellular signal transduction
	1.6e-04	15	Cellular response to chemical stimulus

STRING-db API access. We need to find the taxonomy id of your species, this used by STRING. First we try to guess the ID based on iDEP’s database. Users can also skip this step and assign NCBI taxonomy id directly by findTaxonomyID.out = 10090 # mouse 10090, human 9606 etc.

 STRING10_species = read.csv(STRING10_speciesFile)  
 ix = grep('Arabidopsis thaliana', STRING10_species$official_name ) 
 findTaxonomyID.out <- STRING10_species[ix,1] # find taxonomyID
 findTaxonomyID.out  

## [1] 3702

Enrichment analysis using STRING

  STRINGdb_geneList.out <- STRINGdb_geneList() #convert gene lists

## Warning:  we couldn't map to STRING 0% of your identifiers

 input_STRINGdbGO <- 'Process'  #'Process', 'Component', 'Function', 'KEGG', 'Pfam', 'InterPro' 
 results <- stringDB_GO_enrichmentData()  # enrichment using STRING 

## Warning in string_db$get_enrichment(ids, category = input_STRINGdbGO, methodMT =
## "fdr", : methodMT parameter is depecated. Only FDR correction is available.

## Warning in string_db$get_enrichment(ids, category = input_STRINGdbGO, methodMT =
## "fdr", : iea parameter is deprecated.

## [1] "Process"

## Warning in string_db$get_enrichment(ids, category = input_STRINGdbGO, methodMT =
## "fdr", : methodMT parameter is depecated. Only FDR correction is available.

## Warning in string_db$get_enrichment(ids, category = input_STRINGdbGO, methodMT =
## "fdr", : iea parameter is deprecated.

## [1] "Process"

 results$adj.Pval <- format( results$adj.Pval,digits=3 )
 kable( results, row.names=FALSE) %>%
  kable_styling(bootstrap_options = c("striped", "hover")) %>%
  scroll_box(width = "100%") 

“No significant enrichment found.”	adj.Pval
No significant enrichment found.	NULL

PPI network retrieval and analysis

 input_nGenesPPI <- 100 #Number of top genes for PPI retrieval and analysis 
 stringDB_network1(1) #Show PPI network 

Generating interactive PPI

 write(stringDB_network_link(), 'PPI_results.html') # write results to html file 

## Warning: 'string_db$get_link' is deprecated.
## Use 'Contact developers to request functionality' instead.
## See help("Deprecated")

## Warning:  we couldn't map to STRING 0% of your identifiers

## Warning: 'string_db$get_link' is deprecated.
## Use 'Contact developers to request functionality' instead.
## See help("Deprecated")

## Warning: 'string_db$get_link' is deprecated.
## Use 'Contact developers to request functionality' instead.
## See help("Deprecated")

 browseURL('PPI_results.html') # open in browser 

8. Pathway analysis

 input_selectContrast1 <- 'Microgravity-Terrestrial'    #select Comparison 
 #input_selectContrast1 = limma.out$comparisons[3] # manually set
 input_selectGO <- 'GOBP'   #Gene set category 
 #input_selectGO='custom' # if custom gmt file
 input_minSetSize <- 15 #Min size for gene set
 input_maxSetSize <- 2000   #Max size for gene set 
 # Read pathway data again 
 GeneSets.out <-readGeneSets( geneSetFile,
    convertedData.out, input_selectGO,input_selectOrg,
    c(input_minSetSize, input_maxSetSize)  ) 
 input_pathwayPvalCutoff <- 0.2 #FDR cutoff
 input_nPathwayShow <- 30   #Top pathways to show
 input_absoluteFold <- FALSE    #Use absolute values of fold-change?
 input_GenePvalCutoff <- 1  #FDR to remove genes 

 input_pathwayMethod = 1  # 1  GAGE
 gagePathwayData.out <- gagePathwayData()  # pathway analysis using GAGE  
   
 results <- gagePathwayData.out  #Enrichment analysis for k-Means clusters  
 results$adj.Pval <- format( results$adj.Pval,digits=3 )
 kable( results, row.names=FALSE) %>%
  kable_styling(bootstrap_options = c("striped", "hover")) %>%
  scroll_box(width = "100%") 

Direction	GAGE analysis: Microgravity vs Terrestrial	statistic	Genes	adj.Pval
Down	Photosynthesis	-5.401	223	1.1e-04
	Ribonucleoprotein complex biogenesis	-5.01	438	3.5e-04
	RNA modification	-4.8591	321	4.9e-04
	Ribosome biogenesis	-4.734	343	7.0e-04
	NcRNA metabolic process	-4.2086	425	5.6e-03
	NcRNA processing	-4.1143	357	7.2e-03
	Cellular response to DNA damage stimulus	-3.7807	337	2.3e-02
	RRNA processing	-3.7162	239	2.5e-02
	DNA repair	-3.7091	314	2.5e-02
	RRNA metabolic process	-3.6563	244	2.8e-02
Up	Cellular response to decreased oxygen levels	7.4246	176	3.4e-10
	Cellular response to oxygen levels	7.4246	176	3.4e-10
	Cellular response to hypoxia	7.3912	175	3.4e-10
	Response to hypoxia	7.0149	197	1.8e-09
	Response to oxygen levels	6.991	201	1.8e-09
	Response to decreased oxygen levels	6.9904	200	1.8e-09
	Root epidermal cell differentiation	4.1893	115	5.4e-03
	Plant epidermal cell differentiation	4.0288	132	7.7e-03
	Response to toxic substance	4.0187	275	7.7e-03
	Trichoblast differentiation	3.938	102	1.1e-02
	Cell maturation	3.6762	96	2.2e-02
	Trichoblast maturation	3.6762	96	2.2e-02
	Root hair cell differentiation	3.6762	96	2.2e-02
	Anatomical structure maturation	3.5015	151	3.2e-02
	Response to antibiotic	3.4886	255	3.2e-02
	Antibiotic metabolic process	3.4875	188	3.2e-02
	Antibiotic catabolic process	3.455	78	3.8e-02
	Response to drug	3.4042	482	3.8e-02
	Cellular response to toxic substance	3.3863	148	3.8e-02
	Response to reactive oxygen species	3.3836	145	3.8e-02

 pathwayListData.out = pathwayListData() 
 enrichmentPlot(pathwayListData.out, 25  ) 

  enrichmentNetwork(pathwayListData.out )  

  enrichmentNetworkPlotly(pathwayListData.out) 

## Warning: `arrange_()` is deprecated as of dplyr 0.7.0.
## Please use `arrange()` instead.
## See vignette('programming') for more help
## This warning is displayed once every 8 hours.
## Call `lifecycle::last_warnings()` to see where this warning was generated.

 input_pathwayMethod = 3  # 1  fgsea 
 fgseaPathwayData.out <- fgseaPathwayData() #Pathway analysis using fgsea 

## Warning in fgsea(pathways = gmt, stats = fold, minSize = input_minSetSize, :
## You are trying to run fgseaSimple. It is recommended to use fgseaMultilevel. To
## run fgseaMultilevel, you need to remove the nperm argument in the fgsea function
## call.

 results <- fgseaPathwayData.out  #Enrichment analysis for k-Means clusters 
 results$adj.Pval <- format( results$adj.Pval,digits=3 )
 kable( results, row.names=FALSE) %>%
  kable_styling(bootstrap_options = c("striped", "hover")) %>%
  scroll_box(width = "100%") 

Direction	GSEA analysis: Microgravity vs Terrestrial	NES	Genes	adj.Pval
Down	Photosynthesis	-2.3258	223	6.5e-03
	Flavonoid metabolic process	-2.3107	68	6.5e-03
	Response to UV-B	-2.2799	59	6.5e-03
	Flavonoid biosynthetic process	-2.2798	50	6.5e-03
	Response to UV	-2.1905	95	6.5e-03
	Regulation of flavonoid biosynthetic process	-2.1518	16	6.5e-03
	Regulation of anthocyanin metabolic process	-2.0566	19	6.5e-03
	Response to gravity	-2.0493	78	1.0e-02
	Photosynthesis, light reaction	-2.011	119	6.5e-03
	Anthocyanin-containing compound biosynthetic process	-1.9906	20	1.4e-02
	Anthocyanin-containing compound metabolic process	-1.9898	35	1.4e-02
	Regulation of response to red or far red light	-1.9835	34	1.6e-02
	Response to far red light	-1.9802	45	1.0e-02
	Response to red light	-1.9735	55	1.6e-02
Up	Cellular response to decreased oxygen levels	2.4397	176	6.5e-03
	Cellular response to oxygen levels	2.4397	176	6.5e-03
	Cellular response to hypoxia	2.4371	175	6.5e-03
	Response to hypoxia	2.3544	197	6.5e-03
	Response to decreased oxygen levels	2.3491	200	6.5e-03
	Response to oxygen levels	2.3466	201	6.5e-03
	Root epidermal cell differentiation	2.2177	115	6.5e-03
	Trichoblast differentiation	2.2171	102	6.5e-03
	Plant epidermal cell differentiation	2.1658	132	6.5e-03
	Cell maturation	2.1644	96	6.5e-03
	Trichoblast maturation	2.1644	96	6.5e-03
	Root hair cell differentiation	2.1644	96	6.5e-03
	Root hair cell development	2.1025	78	6.5e-03
	Root hair elongation	2.0496	58	6.5e-03
	Response to hydrogen peroxide	2.0103	65	6.5e-03
	Indole glucosinolate metabolic process	1.9891	27	9.8e-03

  pathwayListData.out = pathwayListData() 
 enrichmentPlot(pathwayListData.out, 25  ) 

  enrichmentNetwork(pathwayListData.out )  

  enrichmentNetworkPlotly(pathwayListData.out) 

   PGSEAplot() # pathway analysis using PGSEA 

## 
## Computing P values using ANOVA

9. Chromosome

 input_selectContrast2 <- 'Terrestrial-Microgravity'    #select Comparison 
 #input_selectContrast2 = limma.out$comparisons[3] # manually set
 input_limmaPvalViz <- 0.1  #FDR to filter genes
 input_limmaFCViz <- 2  #FDR to filter genes 
 genomePlotly() # shows fold-changes on the genome 

## Warning in eval(quote(list(...)), env): NAs introduced by coercion

## Warning in genomePlotly(): NAs introduced by coercion

10. Biclustering

 input_nGenesBiclust <- 1000    #Top genes for biclustering
 input_biclustMethod <- 'BCCC()'    #Method: 'BCCC', 'QUBIC', 'runibic' ... 
 biclustering.out = biclustering()  # run analysis

 input_selectBicluster <- 1 #select a cluster 
 biclustHeatmap()   # heatmap for selected cluster 

 input_selectGO4 <- 'GOBP'  #Gene set category 
 # Read pathway data again 
 GeneSets.out <-readGeneSets( geneSetFile,
    convertedData.out, input_selectGO4,input_selectOrg,
    c(input_minSetSize, input_maxSetSize)  )  
 results <- geneListBclustGO()  #Enrichment analysis for k-Means clusters   
 results$adj.Pval <- format( results$adj.Pval,digits=3 )
 kable( results, row.names=FALSE) %>%
  kable_styling(bootstrap_options = c("striped", "hover")) %>%
  scroll_box(width = "100%") 

adj.Pval	Genes	Pathways
1.9e-120	192	Peptide metabolic process
1.9e-117	182	Translation
3.3e-117	182	Peptide biosynthetic process
8.8e-113	185	Amide biosynthetic process
2.1e-111	196	Cellular amide metabolic process
5.5e-96	238	Organonitrogen compound biosynthetic process
1.4e-68	151	Response to inorganic substance
6.0e-57	101	Response to metal ion
1.3e-53	86	Response to cadmium ion
4.4e-49	197	Response to abiotic stimulus

11. Co-expression network

 input_mySoftPower <- 5 #SoftPower to cutoff
 input_nGenesNetwork <- 1000    #Number of top genes
 input_minModuleSize <- 20  #Module size minimum 
 wgcna.out = wgcna()   # run WGCNA  

## Warning: executing %dopar% sequentially: no parallel backend registered

##    Power SFT.R.sq  slope truncated.R.sq mean.k. median.k. max.k.
## 1      1   0.7780  1.540          0.918  368.00    378.00  520.0
## 2      2   0.2370  0.285          0.681  189.00    190.00  333.0
## 3      3   0.0936 -0.126          0.651  114.00    108.00  234.0
## 4      4   0.5360 -0.387          0.797   74.60     67.20  176.0
## 5      5   0.7270 -0.553          0.905   52.10     44.20  140.0
## 6      6   0.8430 -0.724          0.971   38.10     30.50  116.0
## 7      7   0.8550 -0.824          0.955   28.80     21.40   98.1
## 8      8   0.9000 -0.922          0.984   22.40     15.90   84.7
## 9      9   0.9090 -0.995          0.969   17.80     12.00   74.2
## 10    10   0.9080 -1.060          0.953   14.40      9.25   65.6
## 11    12   0.9290 -1.130          0.950    9.86      5.67   52.5
## 12    14   0.9370 -1.180          0.941    7.06      3.53   43.0
## 13    16   0.9550 -1.210          0.949    5.24      2.34   35.8
## 14    18   0.9530 -1.220          0.940    4.00      1.54   30.2
## 15    20   0.9730 -1.230          0.965    3.12      1.08   25.8
## TOM calculation: adjacency..
## ..will not use multithreading.
##  Fraction of slow calculations: 0.000000
## ..connectivity..
## ..matrix multiplication (system BLAS)..
## ..normalization..
## ..done.

 softPower()  # soft power curve 

  modulePlot()  # plot modules  

  listWGCNA.Modules.out = listWGCNA.Modules() #modules

 input_selectGO5 <- 'GOBP'  #Gene set category 
 # Read pathway data again 
 GeneSets.out <-readGeneSets( geneSetFile,
    convertedData.out, input_selectGO5,input_selectOrg,
    c(input_minSetSize, input_maxSetSize)  ) 
 input_selectWGCNA.Module <- 'Entire network'   #Select a module
 input_topGenesNetwork <- 10    #SoftPower to cutoff
 input_edgeThreshold <- 0.4 #Number of top genes 
 moduleNetwork()    # show network of top genes in selected module

##  softConnectivity: FYI: connecitivty of genes with less than 12 valid samples will be returned as NA.
##  ..calculating connectivities..

 input_removeRedudantSets <- TRUE   #Remove redundant gene sets 
 results <- networkModuleGO()  #Enrichment analysis of selected module
 results$adj.Pval <- format( results$adj.Pval,digits=3 )
 kable( results, row.names=FALSE) %>%
  kable_styling(bootstrap_options = c("striped", "hover")) %>%
  scroll_box(width = "100%") 

adj.Pval	Genes	Pathways
1.9e-120	192	Peptide metabolic process
1.9e-117	182	Translation
3.3e-117	182	Peptide biosynthetic process
8.8e-113	185	Amide biosynthetic process
2.1e-111	196	Cellular amide metabolic process
5.5e-96	238	Organonitrogen compound biosynthetic process
1.4e-68	151	Response to inorganic substance
6.0e-57	101	Response to metal ion
1.3e-53	86	Response to cadmium ion
4.4e-49	197	Response to abiotic stimulus